ES2414605T3 - Ubiquitin or crystalline gamma conjugates for use in therapy, diagnosis, and chromatography - Google Patents

Abstract

Procedure for the preparation of a conjugate, which includes the following components: one or more polypeptide molecules (I) based on human ubiquitin, and, covalently linked with it, one or more functional components (II) that are chosen from the set formed by polypeptides and proteins, organic polymers, sugars, low molecular weight substances, peptides, as well as derivatives of these substances, realizing that, after the coupling of (I) to (II), the functionality of all the components remains preserved, and realizing that the procedure comprises the following steps: a) Provision of human ubiquitin; b) Provision of a ligand, which is chosen from the set consisting of some (poly) peptides / proteins; c) Selection of amino acids 2, 4, 6, 62, 63, 64, 65 and 66 of human ubiquitin; d) Modification of the selected amino acids by substitution mediating conservation of the ubiquitin-like folding pattern; e) Contacting the modified protein with the ligand made available in step b); f) Determination of those proteins, which are suitable for a specific binding to ligands with a KD dissociation constant> = 10-5 M or less, detecting binding activity using the ELISA technique or a MRI superficial plasmons; g) Coupling of components (I) and (I) in a region located outside the superficially exposed region of the ß-folded sheet of the polypeptide molecule (I), which is intended for specific binding to the ligand, through terminal fusion of peptides with (I), or through cysteine or lysine side chains in (I); h) Isolation of the conjugate; and i) Detection of the functionality of both components of the conjugate.

Description

Ubiquitin or crystalline gamma conjugates for use in therapy, diagnosis, and chromatography

The present invention relates to a process for the preparation of a conjugate comprising one or more polypeptide molecules based on human ubiquitin and one or more functional component parts.

International patent application document WO 01/04144 A discloses conjugates of a protein with a beta folded sheet structure (crystalline gamma mutant), which binds to estradiol. In particular, the terminal fusion of crystalline gamma mutants with various molecules, namely with an E-label, with minority coat protein 3 as well as with His-label is described.

WO 01/04144 A further describes the process of modifying stable beta-folded sheet proteins by site-specific mutagenesis in the beta-folded sheet next to the surface, such that a non-binding protein results protein with specific fixing properties.

Human ubiquitin finds use as a scaffold protein for the generation of alternative binding molecules. Ubiquitin is a small, monomeric and cytosolic protein, which occurs - largely preserved in its sequence - in all known eukaryotic cells, from those of protozoa to vertebrates. It plays a fundamental role in the body in the regulation of the controlled degradation of the cell's own proteins.

The ubiquitin polypeptide chain is composed of 76 amino acids, which are folded into an extremely compact alpha / beta structure (Vijay-Kumar, 1987): Almost 87% of the polypeptide chain participates by means of hydrogen bridges in the formation of the secondary structural elements. As prominent secondary structures there may be valid three and a half coils in the form of an alpha helix as well as an antiparallel beta folded sheet, which is composed of five chains. The characteristic arrangement of these elements - an antiparallel beta folded sheet, exposed towards the surface of the protein, which is covered on its posterior side by an alpha helix that is vertically above it - is generally valid as a so-called motif of folding of the type of the ubiquitin. Another feature of the structure is a pronounced hydrophobic region within the protein between the alpha helix and the beta folded sheet.

The artificial production of ubiquitin is possible, due to its small size, both by chemical synthesis as well as by biotechnological procedures. Due to the favorable folding properties, ubiquitin can be produced in the case of obtaining by genetic technology with the help of microorganisms such as E. coli in relatively large quantities, optionally in the cytosol or in the enclosure periplasmic.

The simple and efficient bacterial production makes it possible to use ubiquitin as a participant in a fusion for other foreign proteins that must be produced, whose production is problematic. By fusion with ubiquitin, improved solubility and thus improved performance can be achieved (Butt et al., 1989).

Based on the existing data on crystal structures present (registration in the PDB data bank: 1UBI), by means of a computer-supported analysis, the positions of such amino acids in the ubiquitin protein framework, whose side chains are exposed on the surface, that is to say that they are oriented towards the solvent or a potential participant in the fixation. The chosen positions are in spatial proximity to each other next to the beginning of the first terminal chain of the beta folded foil (positions 2, 4, 6) as well as in the loop (positions 62, 63) or respectively next to the beginning of the carboxy terminal chain of the beta folded sheet (positions 64, 65, 66) and form with their amino acid side chains a coherent region on the surface of ubiquitin. By means of random amino acid substitutions in the analyzed region, a hypervariable, surface exposed region was thus generated in the ubiquitin protein structure that remains intact (see European PCT patent document PCT / EP2004 / 005730, unpublished ).

The generation of an artificial fixation surface on a beta folded sheet protein constitutes an interesting new alternative to the usual antibodies. Demonstration could be provided that a new artificially generated binding site on the surface of crystalline or ubiquitin-like gamma proteins leads to binding molecules capable of function (see German patent application document DE 19932688 A1) (see document PCT / EP2004 / 005730, unpublished).

Until now, however, any incitement or mention about the possibility of coupling these polypeptide molecules to an additional component for the formation of a conjugate was lacking, in order to make them useful for diagnostic, therapeutic and analytical uses, without in this case suffering a loss function of one of the two components or both.

The present invention is therefore based on the mission of making available a process for the production of a conjugate based on a ubiquitin-based polypeptide and a functional component, the respective polypeptide molecule having a modified binding property compared to the wild-type polypeptide for specific binding to a ligand, with both components of the conjugate being realized, after coupling to each other, to retain their functionality or to be further enhanced by coupling.

The problem posed by this mission is solved by the object of the independent claims. Preferred embodiments are set out from the dependent claims.

The present invention relates to the selective, site-specific, non-targeted coupling of novel ubiquitin-based binding proteins to the most diverse molecules, such as eg proteins (chromophoric proteins and enzymes). ), matrices (eg dextran, a polymethacrylate, an agarose, a Sepharose, derivatives of a polystyrene and a cellulose) and small molecules (eg, fluorescence markers, biotin, digoxigenin, radioisotopes, antibiotics, among others). These molecules, called AffilinTM, have in common the "de novo" design of a binding region in the beta-pleated sheet structures of proteins. Consequently, they differ from the most prominent class of binding proteins in nature, that of antibodies, in that the binding region is located in flexible loop regions (the CDR's, short for Complementary Determining Regions = regions determinants of protein). Another difference between AffilinTM and antibodies is the size of the molecules. The polypeptides used as the basis for the first component of the conjugate according to the invention have a molecular weight of 10 kDa (ubiquitin), and the antibodies have a molecular weight of 150 kDa (IgG).

The present invention encompasses a process for the preparation of conjugates based on these polypeptides and of the corresponding partners in the coupling. Furthermore, their use in various uses has been described and it has surprisingly been shown that these coupling methods lead neither to loss of biological activity of the coupling partner nor to loss of binding properties of polypeptides to its ligands.

By way of example for the invention, the coupling of the polypeptide components to a dextran matrix in the BIACORE system, the coupling to the Oyster®556 fluorescent dye, to the chromophore protein Phycoerythrin (PE) and to the horseradish peroxidase enzyme are described. rusticano, as well as the immobilization of a polypeptide to a support material for use in affinity chromatography. In accordance with the invention, couplings in this case were made either directly to the polypeptide molecule, eg to nucleophilic side chains of proteins, or in a target-directed manner, to peptide linkers at the terminal end of C. Surprisingly, the coupling methods were transferable to both frameworks, and did not lead to any prejudice to the binding properties of these molecules. The coupling of the relatively small (10 kDa) polypeptide molecules with large proteins such as eg a phycoerythrin (240 kDa) could not be expected, with a simultaneous preservation of binding activity.

The conjugates obtained in this way, surprisingly, do not restrict the binding capacity of the AffilinTM molecules, but rather, in the case of certain conjugates, an increase in the macroscopic dissociation constant (avidity effect) could be observed, which offers other possibilities for the use of polypeptide molecules, eg, in therapy. Furthermore, it was shown that these polypeptide molecules show binding activity also after coupling to water-insoluble matrices, and that they are regenerable after treatment with denaturing agents such as urea, guanidinium, ethanol, bleach. soda or hydrochloric acid, that is, its fixing properties can be restored. By means of a detailed analysis of the structure data of some ubiquitin-based molecules, the possibility of selectively structuring the non-specific coupling was offered, by using deliberately selected lysine residues. Structural analysis showed that lysines are, in the case of crystalline gamma, in the C-terminus domain of the protein and therefore should be suitable for coupling (see Figure 1). Also in the case of ubiquitin, the identification of such lysine residues was achieved. However, for such coupling strategies it must be ensured that no other lysine is found in the binding region.

For the present invention, an NGF-binding polypeptide was selected from the human ubiquitin library (UB10) and then purified. By introducing a special peptide linker at the C-terminus with a defined length, including a cysteine, it was possible to modify the polypeptide molecules in such a way that a selective coupling was obtained without any damage to the binding activity. For effective coupling, it is necessary to remove all remaining accessible cysteines. In the case of the unspecific coupling method of the polypeptide molecules, it was surprisingly successful both to preserve the binding activity of the polypeptides and, in addition to this, to achieve an increase in affinity by avidity, and therefore to prepare a very attractive conjugate of molecules based on in polypeptides for diagnosis and therapy.

Such alternative binding molecules can be universally used in therapy, diagnosis, and chromatography. By coupling polypeptides to the most diverse participants, an even wider sector of use is opened for these new binding molecules.

The present invention encompasses the following aspects and embodiments:

In accordance with a first aspect, the present invention relates to a process for the preparation of a conjugate comprising the following components: one or more polypeptide molecules (I) based on human ubiquitin, and, united with them by covalent bonds, one or more functional components (II) that are chosen from the set consisting of polypeptides and proteins, organic polymers, sugars, low molecular weight substances, peptides, as well as derivatives of these substances, realizing that, after the coupling of

(I) to (II) the functionality of all the components remains conserved, and realizing that the process comprises the following steps: a) making human ubiquitin available; b) making available a ligand, which is chosen from the set consisting of some (poly) peptides / proteins; c) selection of amino acids 2, 4, 6, 62, 63, 64, 65 and 66 of human ubiquitin; d) modification of selected amino acids by substitution mediating preservation of the ubiquitin-like folding motif; e) contacting the modified protein with the ligand made available in step b); f) determination of those proteins, which are suitable for specific binding to ligands with a dissociation constant of KD = 10-5 M or less, detecting binding activity using the ELISA technique or a surface plasmon resonance; g) coupling of components (I) and (II) in a region located outside the superficially exposed region of the folded sheet � of the polypeptide molecule (I), which is intended for specific binding to the ligand, through a terminal peptide fusion to (I), or through cysteine or lysine side chains in (I); h) isolation of the conjugate; and i) detection of the functionality of both components of the conjugate.

In other words, the conjugate used according to the invention does not encompass ubiquitin in its wild-type form, but only in a form adapted to the special ligands. This specially adapted form provides for binding properties to the respective ligands, modified (improved) or generated in new compared to the wild type form. As a common principle of ubiquitin-based polypeptide molecules, it should be mentioned that the generation of an artificial binding surface is carried out on a beta folded sheet structure, in order to make possible a specific binding to a ligand of interest. The presence of at least one beta folded sheet structure for the provision of an artificial fixing surface is therefore an essential feature of the invention.

A conjugate prepared according to the invention in this context designates the post-translational covalent bonding of a polypeptide molecule with another component and is therefore distinguished eg from a fusion of polypeptides on the genetic level. The fused polypeptides are the result of a so-called translational fusion.

The ubiquitin-based polypeptide molecule is preferably chosen according to the invention from the set consisting of proteins from the ubiquitin-like proteins superfamily of proteins, proteins that have a folding motif similar to that of ubiquitin, as well as fragments or fusion proteins of these, which in each case have a folding motif similar to that of ubiquitin, being that the protein, mediating conservation of the folding motif similar to that of ubiquitin, due to one or more amino acid modifications in at least one exposed region on the surface, including at least one beta folded foil chain from the beta folded foil region and optionally non-foil regions beta-folded, has a binding affinity, which was not previously present, against a pre-established binding partner or respectively lig I'm going.

Accordingly, we also describe a protein modified by a substitution, which is chosen from the set consisting of the proteins of the superfamily of "ubiquitin-like proteins", which have a folding motif similar to that of ubiquitin, as well as fragments or fusion proteins of these, which all have in each case a folding motif similar to that of ubiquitin, the protein owning, due to this modification, a binding affinity, that was not present before, in front of a pre-established participant in the fixation.

The ubiquitin-based polypeptide molecule (I), which is used in the conjugate according to the invention, is accordingly prepared by a modification of human ubiquitin. The modifications cover in particular the amino acid exchange. These modifications are carried out in at least one exposed region on the surface of the protein that must be modified. The modification of at least one amino acid encompasses at least one beta folded foil chain from the beta folded foil region, the beta folded foil chain must be located next to the surface of the protein such that it is accessible to the binding partner or respectively the ligand, which can be bound with a determinable affinity to the modified protein. In another embodiment of the invention, in addition to the modifications in the beta folded sheet chain of the beta folded sheet region, non-beta folded sheet regions are also modified, which are preferably exposed on the surface, in order to influence the affinity of

binding against the pre-established binding partner, in particular in order to increase it and consequently to increase specificity.

For a person skilled in the art, the most diverse techniques, which are known per se, are available for modifying one or more amino acid (s). These are described in more detail below. In a complementary way, mention is also made of the publications of Ausuebel et al., 1994, as well as Sambrook et al., 1989.

Certain amino acid modifications of the core region of ubiquitin, which is not exposed on the surface, are already known (see citations from Finucane et al., 1999; and from Lazar et al., 1997). The modifications that have been carried out there concern positions that, due to their location in the hydrophobic core, do not participate in the fixings, since they are not accessible to the solvent or the possible participants in the fixation.

Hereinafter, what is understood in this invention is to be illustrated by the concept of "a fixation property, which was not previously present" or respectively "an artificial fixation site, generated de novo", or respectively "a property of modified binding compared to wild-type polypeptide for specific binding to a ligand. " By this concept it is understood that the modified protein previously did not have, next to the modified region, any binding property for a participant in the pre-established fixation or for a participant in the natural fixation of ubiquitin.

Binding partners, which can also be defined as ligands, have a measurable affinity for the modified protein according to the invention. As a minimum value for the presence of a quantifiable binding property, that is, the affinity with which the participant is bound, a dissociation constant for the formed complex of KD = 10-5 M or more can be considered according to the invention little. From a value of 10-15 M it is possible to start from a quantifiable binding affinity. Depending on the use, a value of from 10-6 M to 10-12 M is preferred, even more preferably from 10-6 to 10-11 M for eg chromatographic uses, or from 10-9 up to 10-12 M for eg diagnostic or therapeutic uses. Other preferred binding affinities are in the region of from 10-7 to 10-10 M, preferably up to 10-11 M. The procedures for determining binding affinities are known per se and are described in the following pages.

According to the invention, the term "modification" is understood to mean amino acid replacements.

According to the invention, the superfamily of ubiquitin-like proteins encompasses the subgroups listed in the quote by Murzin et al. (1995). These include, for example, families of "ubiquitinrelated proteins", "UBX domain", "GABARAP-like", "RAS-binding domain "(from RAS fixing domain) etc. Proteins, which have a folding motif similar to that of ubiquitin, are also encompassed. Examples of these are SUMO-1, FAU, NEDD-8, UBL-1, and GDX, as well as Rub1, APG8, ISG15, URM1, HUB1, Elongin B, PLIC2 (the domain of the N-terminus), and human Parkin ( the domain of the terminal end of N).

The proteins in the superfamily of ubiquitin-like proteins have been defined very precisely. By way of example only, reference is made to the Internet page: http://bip.weizmann.ac.il/scop/index.html. According to this, the family of ubiquitin-like proteins is defined as a superfamily, to which the family of ubiquitin-related proteins belongs. All the members of this superfamily are distinguished predominantly by antiparallel arranged folded sheets, which are subdivided into segments a and �. Folding is defined as beta-Grasp and therefore similar to ubiquitin. The nucleus region is defined as follows: beta (2) -alpha-beta (2), the numbers denoting the number of the chains, realizing that the sum of the chains forms the folded sheet �. The arrangement of the mixed beta folded foil is 2143, which designates the location of the chains in the case of inspection from left to right on the beta folded foil (the terminal end of amino is below, and the end carboxy terminal is located above). For members of ubiquitin-like proteins, therefore, a folded � antiparallel sheet exposed to the surface of the protein is characteristic, which is covered on its posterior side by a helix a that is located vertically above it. This ubiquitin-like folding motif is characteristic for proteins and unequivocally distinguishes family members from other proteins. Taking this definition into account, the N-terminal domain of PLIC-2 similar to that of ubiquitin and the Parkin domain similar to that of ubiquitin are also encompassed.

A person skilled in the art, or with the help of sequence comparisons, the so-called alignments, or structural overlaps, can draw some initial conclusions about whether the protein is a member of the superfamily of ubiquitin-like proteins. Definitive safety is always naturally provided by a structural analysis, for example a structural analysis by X-ray crystallography or multidimensional nuclear resonance spectroscopy. In the meantime, good predictions can also be achieved through structural analysis with the help of genetic algorithms.

Other information about the ubiquitin superfamily is found, for example, in the publication by Larsen et al., 2002. In a complementary way, mention should also be made of the publication by Buchberger et al. 2001. Buchberger describes the typical folding �- Grasp as a characteristic of ubiquitin-like proteins with a secondary structure with the organization beta-beta-alpha-beta-beta-alpha-beta, ie an arrangement of five beta chains in the form of a mixed sheet (in English "mixed- sheet ") in provision 21534. In this context it is noteworthy that the UBX does not have any significant homology in the primary sequence with eg ubiquitin (Buchberger et al., 2001), but nevertheless - due to its three-dimensional structure, which is identical to that of eg ubiquitin - it is classified within ubiquitin-like proteins. In this connection, it should be mentioned that, in ubiquitin, amino acids located at positions 48 and 49 are also considered in part as an autonomous beta chain (Vijay-Kumar, 1987). This fifth chain, which would be located in the ubiquitin structure behind the helix, and which would provide the mixed plate with arrangement 21534, has however only two amino acids, and it is quite doubtful to designate this chain that consists of two amino acids. like a beta folded foil chain. However, according to Buchberger et al. (2001), as discussed above, also proteins with arrangement 21534 could be subordinated without commitment within the superfamily of ubiquitin-like proteins. For the present invention, as regards the arrangement of beta chains in ubiquitin the definition of 21543 described in detail above was chosen.

The mentioned family and superfamily proteins are generally highly conserved. For example, according to current knowledge, ubiquitin has the same amino acid sequence in all mammals. Yeast ubiquitin has only a three amino acid deviation from it. Human ubiquitin or respectively mammalian ubiquitin is made up of 76 amino acids and has the structure described above.

The modified protein used according to the invention should have an amino acid sequence match of at least 30%, preferably at least 40% or 50%, even more preferably at least 60%, of at least 70%, of at least 80%, of at least 90%, or of at least 95%, with respect to the starting protein, which is modified, is say with respect to human ubiquitin, the protein having in each case a folding motif of the ubiquitin type, as defined in detail above.

According to the present invention, the protein, which is chosen for the production of the modified protein, is human ubiquitin. If the sector of use of the proteins produced according to the invention is known, that is to say that if for example the modified protein is to be used as a medicine for the treatment of diseases in human beings, a human protein can preferably be used as the starting protein that must be modified; this is similarly valid for the corresponding employment sectors.

Human or mammalian ubiquitin respectively has 76 amino acids. The amino acids of the four beta chains, which contribute to the formation of the antiparallel beta folded sheet, are, according to the invention, corresponding to the 1UBQ structure found in the PDB database (see the internet page http: // www.rcsb.org/pdb/index.html), the following amino acid positions:

The first chain (at the amino terminus): 2 to 7; the second beta folded foil chain: 12 to 16; the third chain: 41 to 45; the fourth chain (at the terminal end of carboxy): 66 to 71. The situation of the chains in the case of inspection from left to right on the folded sheet (the terminal end of amino is below, the terminal end of carboxy found above) is: 2nd, 1st, 4th, 3rd, strand, the polypeptide strand between the 1st and 4th strand forming an alpha helix.

Selection and modification of the amino acids that must be modified:

Based on corresponding structure data, such as is freely available eg in the Protein Data BankTM (Berman et al., 2000; http://www.rcsb.org/pdb), through a computer-supported analysis The positions of those amino acids in the starting protein can be located, eg in the protein framework of ubiquitin, whose side chains are exposed on the surface, that is, they are oriented towards the solvent or a potential participant in fixation . Furthermore, by means of a computer-supported analysis, those amino acids can be identified in the starting protein, that is, in ubiquitin, whose random replacement presumably could not have any effect or could only have a small negative effect on the stability of the protein framework.

This information may represent a first starting point for the suitability of each individual amino acid as an element of a binding site, which then requires verification in practice. According to the invention, due to its exposure on the surface and the tolerance of the total structure against its random exchange, the amino acids located at positions 2, 4, 6, 62, 63, 64, 65 and 66 in ubiquitin were chosen human.

Said positions are in spatial proximity to each other at the beginning of the first beta-folded foil chain at the amino terminus (positions 2, 4, 6) as well as in the loop (positions 62, 63)

or respectively at the beginning of the beta folded foil chain at the carboxy terminus (positions 64, 65, 66) and form with their amino acid side chains a coherent region on the surface of ubiquitin (see Fig. 1). By means of random amino acid substitutions ("randomization") in the analyzed region - analogous to the antigen binding site of the antibodies - in the protein structure, a hypervariable region exposed on the surface, still intact, of ubiquitin can be generated .

With the help of the ProSAII software ("Protein Structure Analysis"; from Proceryon Biosciences, Salzburg) it was possible to determine, for example, the protein stability of 104 variants compared to ubiquitin (WT = wild type ) and an equally large random sample of some variants, in which the residues of a "control epitope" were replaced (randomized positions 24, 28, 31, 32, 35, 37, 38, 39). In this case, approximately 19% of the variants generated in silico (by computer) that had been randomly replaced in the region of the binding site, have a stability at least as high as that of ubiquitin (WT), while that approximately 90% of them were more stable than the carrier of the "control epitope" (see Fig. 2). This computer-supported result can then serve as a clue to the choice of suitable amino acids.

Preferably - based on the available data on human ubiquitin structures - eight amino acid positions were first chosen in the region of the binding site to be generated. By means of random modifications of the primary sequence in this region (by means of random mutagenesis) and a subsequent specific choice (selection) those variants were obtained, which have the desired binding activity for a pre-established hapten or antigen, or respectively which generally have a participant in the preset fixation. Despite the fact that the modified proteins obtained are thus conferred a de novo binding property, as far as the structure and chemical properties of the proteins are concerned, they remain very broadly identical to the starting protein. In this way, they have advantages such as eg small size, high stability, cheap production, as well as simple modifiability, paired with high affinity and specificity for a previously defined ligand. The suitability of ubiquitin as a framework structure for the generation of artificial binding proteins was not predictable in this case, since 1.) the tolerance of the framework against extensive amino acid exchanges could not be expected due to the small size of the ubiquitin and 2.) the functionality of the artificial fixation site does not appear to be presented beforehand by mediating inclusion of the beta-folded lamina, which is considered to be rigid and inflexible.

Hereinafter, polypeptide molecules based on crystalline gamma (which are not in accordance with the invention) are also described.

Crystalline gamma, a class of crystalline in vertebrates, are monomeric proteins with a molecular mass of approximately 22 kDa. The main structural motif of crystalline gamma is the antiparallel beta folded sheet (Hazes and Hol, 1992, Richardson et al., 1992, Hemmingsen et al., 1994). The crystalline gamma are made up of two very similar globular domains, a domain located at the N-terminus and a domain located at the C-terminus, which are linked to each other by means of a "V" shaped linker peptide. The characteristic folding pattern for crystalline gamma ("Greek Key-Motiv", Slingsby, 1985, Wistow and Piatigorsky, 1988) is quite possibly the cause of the considerable thermal stability as well as stability against denaturing agents (Mandal et al., 1987).

Crystalline gamma II does not normally have any binding properties in the folded state. The modification (by mutagenesis) of a chosen region, exposed to the solvent, of this protein, which is made up of the structural motif of the beta folded sheet, surprisingly led to the modification of the surface structure and the electric charge pattern of the protein and consequently to the production of new fixing properties. In this case, only regions or amino acid positions were chosen, which do not decisively participate in the conservation of the protein structure. Mutagenesis of a small beta-fold lamina protein (Riddle et al., 1997) has shown that certain proteins, despite having considerable sequence modifications, can correctly form, in a high percentage, the natural structure of the folded lamina beta.

In the case of the procedure described here, deliberate mutagenesis is carried out in a protein without any binding properties in the rigid region of the beta-folded sheet. In this way, a protein with considerable stability and with specific binding properties, which is comparable to that of antibody molecules, was produced unexpectedly.

As a suitable system for the isolation of mutagenized beta-pleated sheet proteins with new binding properties, the phage display system ("Phage Display-System") serves. This system enables highly effective screening of a large repertoire of protein variants for specific binding properties (Smith, 1985). In this case, a case is represented in each case

protein variant on the surface of a filamentous phage and this can interact with the target molecules, which are immobilized together with a solid phase. Proteins that bind to the target molecule can be obtained by phage elution. After isolation of phage DNA, the DNA sequence of specifically bound protein variants can be determined. In addition to the phage display system, other selection systems may also find use, such as eg bacteria display (Stahl and Uhlen, 1997) or ribosome display (Hanes et al., 1997).

With the described procedure, it is surprisingly achieved, for example, to modify the beta folded lamina protein, the crystalline gamma II, which is very stable, by means of a deliberate site-specific mutagenesis in the beta folded lamina, next to the surface, such that a protein with specific binding properties results from the non-binding protein. By randomizing eight amino acid positions, therefore, for the first time, mutagenesis is carried out in a framework molecule within a relatively rigid region of the protein. Accordingly, from the crystalline beta gamma II folded sheet protein, a "antibody-like" protein species is produced with respect to its specific binding properties. Crystalline gamma II or other small stable beta-folded sheet proteins can be used generally with the procedure described as new lattice molecules for the design of new binding properties. Modeled beta-folded sheet proteins can replace, for example, recombinant antibodies in various applications.

Additional and more detailed information about this is found in the international patent application document WO 01/04144, which is hereby referenced in its full content.

The concept of "ligand", as defined in the present case, means a substance, which is specifically fixed by a ubiquitin-based polypeptide molecule.

As such binding partners, so-called ligands, some (poly) peptides and proteins (eg immunoglobulins and immunoglobulin derivatives, proteins that can be obtained from blood plasma) are used according to the invention, factors and inhibitors of blood clotting, growth factors, interleukins, cytokines, receptor proteins, viral proteins, and cell surface markers, such as CD14, CD25, CD34), peptides (eg, affinity appendages, such such as the S brand, the T7 brand, the His brand, the Strep brand, the Myc brand, the FLAG brand, and certain peptides of viral origin). About this see also the preferred embodiments, which are set out below.

The concept of "functional component" as used in this context defines the second component of the conjugate, which is attached by covalent bonds to the ubiquitin-based polypeptide molecule. With the expression "functional" it must be stated that it is a suitable and possibly already known component for use in diagnosis, therapy, chromatography and analytics. Expressed in a general way, by the concept of a "functional component" any component is defined with measurable properties, eg an enzymatic activity, a measurable property by spectroscopy, or a toxic property. Apart from this, the structure and function of the functional component in the present conjugate are not restricted. The only requirement is that, after covalent bonding between the polypeptide molecule and the functional component, the functionality of all components remains preserved. In the case of the polypeptide molecule, this functionality is the ability to bind to the special ligand, and in the case of the functional component, for example, its effect as a dye.

In accordance with the invention, one or more than one, or two, functional components may be attached to a polypeptide molecule (as defined above). In order to achieve specific binding of the components to the polypeptide molecule, for example one of the components may be structured for specific binding to a lysine residue, and the other to a cysteine residue in the polypeptide molecule. Examples of two such components are fluorescent dyes, in which case one serves as a fluorescence donor and the other as a fluorescence acceptor.

Some details about the way of fixing the components to each other as well as the fixing components that come into question, are explained below.

Detection of the binding activity of the polypeptide molecule to the ligand and the activity of the partner in the coupling (the functional component) represents - as mentioned above - an important point of view. Detection of ligand binding activity can be carried out in this case by various methods. In the ELISA technique (acronym for "enzyme-linked immunosorbent assay" = enzyme-linked immunosorbent assay), binding is detected by antibody and peroxidase conjugates, and on the contrary, the Biacore system exploits the phenomenon of resonance of superficial plasmons for detection. Other techniques, such as fluorescence titration, fluorescence polarization, or fluorescence correlation spectroscopy (FCS), rely on the fluorophore properties of the Affilin ™. In the case of the detection of the activity of the participant in the coupling, that is, the so-called other functional component, the known properties of this are decisive. In this way, chromophore or fluorophore molecules, such as fluorescent dyes or a phycoerythrin, are analyzed through their spectral properties. Nail

Enzymes, such as a peroxidase or an alkaline phosphatase, are tested through their catalytic conversion of model substrates. Other molecules, such as toxins, can be directly tested in cell culture experiments for their biological activity. In the case of radioisotopes, radioactivity can be measured with the help of corresponding counters. For a number of other molecules, such as sugars and nucleic acids, there are commercially available detection reagents. According to one embodiment, - as outlined above - the binding property generated from new or modified compared to wild-type polypeptide is based on one or more amino acid exchanges in an exposed region on the surface of a folded sheet � of the ubiquitin polypeptide molecule (I). In this case, a number of 6-10 is exchanged, preferably 8 amino acids for each polypeptide molecule (I).

It is especially important to mention that the coupling of (I) to (II) is preferably carried out in a region outside the exposed region on the surface of the folded sheet � of the polypeptide molecule (I), which is intended for specific binding to a ligand. It has been shown that in this way an important requirement is put to the conjugate according to the invention, namely the preservation of the functionality of all the components.

An example will be mentioned here of a coupling to Lys 29, 33 of ubiquitin, which lie outside the binding surface for the ligand, more precisely even outside the beta-pleated lamina (Lys 29, 33 are found on the helix alpha) which is responsible for binding to the ligand. A coupling through this amino acid side chain away from the ligand binding site does not lead to any impairment of the functionality of the conjugate components.

In this sense, it is especially preferred that the coupling of (I) to (II) is carried out in a region located outside the folded sheet � of the polypeptide molecule (I), which has the binding property generated from new or modified for specific binding to a ligand. This region is, in the aforementioned case, eg the alpha helix of the ubiquitin molecule. This is especially evident from Fig. 1, where the domain located at the N-terminus (the beta-pleated sheet) of the crystalline lens has a newly generated binding surface for the ligand, the residues being suitable lysine (highlights) found at the C terminal end portion, for coupling to functional component (II).

According to an embodiment, the coupling or the binding of (I) to / with (II) takes place through amino acid residues of (I). In other words, in this case the coupling is carried out through amino acid residues, which were already present by themselves in the polypeptide molecule (I).

Coupling is effected in this context in a site-specific or non-selectively targeted manner via cysteine or lysine side chains in (I). The concept of "site specific" means, in this context, that a cysteine or lysine is present at or is introduced into a molecule (I) defined site, in order to define an exactly predetermined binding site . The concept of "non-selectively targeted" means that several such residues are present, the attachment to these residues indeed being effected in a selective manner, but being, however, also subject to a certain random factor due to their number.

For the coupling of the two components of the conjugate according to the invention, that is to say for their union by covalent bonds, the following is stated:

Proteins contain a large number of functional groups, through which coupling to other molecules can be achieved. Some concrete examples of them are below. By covalent bonding of the partners, eg via a suitable crosslinking reagent, it is thus possible to bring together various activities in one molecule. By way of example, antibodies will be mentioned in this context, which are frequently coupled with dye molecules and thus provide easily detectable detection reagents, which are then used in diagnosis.

Similarly, certain antibodies are also conjugated to other proteins, preferably with so-called reporter enzymes such as an alkaline phosphatase or a peroxidase. These reporter enzymes chemically convert to a substrate, thereby obtaining a signal detectable by absorption, fluorescence, or luminescence.

By choosing suitable crosslinking reagents, certain proteins, that is to say also the polypeptide molecules according to the invention, can also be coupled to other functional components of many other classes of substances: Thus, for this a large number of polymers come into question. organic and also inorganic, proteins, peptides, nucleic acids, lipids, sugars and also of low molecular weight substances. Preferred partners in the coupling of the polymer class are eg dextran, a polymethacrylate, a Sepharose, an agarose, a polystyrene, a vinyl polymer, a silica gel, a cellulose or a poly (ethylene glycol) (PEG ). The last-mentioned modification is used, for example, for modulation of the pharmacokinetic properties of biotherapeutic agents. A large selection of PEG derivatives, which already

contain pre-activated functional groups, suitable for coupling, are commercially available (from Nektar Therapeutics). The other polymeric materials mentioned serve as vehicle substances in the cases of many biochemical, biotechnological and diagnostic procedures. In this context, chromatographic uses, for example, affinity chromatography, where these polymeric substances find use as a gel matrix, should be highlighted. For this, the surface of a polymeric beads is functionalized and activated with suitable chemical groups, in such a way that the coupling, eg of a protein, with a desired activity is possible. In this way binding proteins with specificity for a certain ligand can also be immobilized and the resulting gel matrix can then be used for efficient and rapid enrichment of the ligand based on a complex mixture of substances. This enrichment can be carried out in this case either by means of column chromatography or also on the surface of filters. Furthermore, for affinity enrichment, it is also possible to use advantageously those polymeric beads that have magnetic properties (magnetic BEADS = magnetic beads) and thus can be effectively separated from a mixture.

An important sector of use is the large-scale technical purification of antibodies for therapeutic purposes from the supernatant material of eukaryotic cell cultures, which is carried out by affinity chromatography with protein A coupled to the matrix, which is a bacterial protein with an intrinsic affinity for antibody molecules.

In the case of the coupling of two protein components, apart from the aforementioned antibody and reporter enzyme fusions, there are numerous examples of use. Thus, in the simplest case, by coupling two or more identical protein molecules, homo-dimers or -multimers can be produced.

In the case of certain binding proteins, avidity effects can also be produced by multimerization, that is, the affinity of the multimer for the target substance is significantly increased compared to the affinity of the monomeric binding protein. By coupling two binding proteins with a specificity for various target substances, on the contrary, so-called bispecific binding molecules can be obtained, which can be used eg as an adapter, in order to bring spatial proximity to respective target substances.

Also some proteins, which possess toxic properties for living cells, the so-called toxins (eg ricin, choleratoxin, the exotoxin of Pseudomonas, among others) are interesting participants in the coupling for antibodies or other binding molecules. After coupling, this bifunctional conjugate can be selectively coupled via the binding protein to a specific target substance eg located on the surface of a tumor cell, and then the affected cell is destroyed by cytotoxic activity.

Other relevant partners in coupling with a protein nature are chromophoric proteins such as eg GFP (green fluorescence protein) and its derivatives, or pigment containing proteins such as phycoerythrins. The resulting coupling products, chromophores or fluorescents, are for their part, as easily detectable substances, valuable tools for research or diagnosis.

However, chromophores or fluorescent protein conjugates, as already mentioned above, are accessible by coupling low molecular weight dye molecules. A large number of suitable dye molecules with crosslinking active groups are commercially available eg from Invitrogen. Other participants in the low molecular weight coupling, which provide protein conjugates for use in diagnosis and therapy, are eg biotin, digoxigenin, heavy metal derivatives, chelating agents, radioisotopes, cytotoxic substances and antibiotics

In the cases of the functional groups contained in proteins and also in the polypeptide molecules according to the invention as well as in functional components, which are suitable for coupling, these are above all amino, carboxy, hydroxy and sulfhydryl groups, but also the function Tyrosine phenolic and aromatic ring systems can serve as attack points for coupling reagents. Suitable amino acid residues for coupling are distinguished by special properties: On the one hand, they have to possess a reactive side chain, which has to be accessible to the coupling reagent. Ideally, this functional group is adjacent to the protein surface and is exposed to the solvent. Furthermore, modification of this residue should not interfere with protein function. Preferably, the residues that must be modified are located at a significant distance from the active center of an enzyme, or from the binding surface of the polypeptide molecule; In addition, no amino acid residues of the same type should be present in these regions, which could lead to a loss of protein function after modification. Therefore, amino acid residues, which rarely occur in the corresponding protein, are also advantageous.

As noted above, for the ubiquitin-based polypeptide (I) molecule, coupling via lysine residues 29 and 33 of the ubiquitin molecule is preferably considered.

Somewhat less preferred, but despite everything possible, is a coupling through lysine residues 11 and 48, which are located in each case at a smaller distance from the ligand-binding surface than residues 29 and 33.

In the case of a crystalline gamma-based polypeptide (I) molecule (which is not according to the invention), the peptide domain with the binding property generated from new or modified compared to wild-type polypeptide, for binding Specific to a ligand, it is the domain located at the N-terminal end, and the coupling of (I) to (II) is carried out through the domain located at the C-terminal end. This also applies to the possibility of a fusion of peptides at the C-terminus, preferably containing cysteine, as well as for amino acid side chains, such as lysine, that are present in the C-terminus domain and are available for a coupling with the functional component. In this case residues 91 and 163 of the crystalline gamma are preferably used (see Fig. 1).

In this case, among the proteinogenic amino acids, lysine, which has an epsilon-amino group in its side chain, as well as cysteine with its sulfhydryl function, should be especially highlighted. These functional groups have a special reactivity and are therefore well suited as partners for a specific coupling of a polypeptide molecule according to the invention with a functional component. Thus, numerous reagents are described in the literature, which react specifically only with sulfhydryl groups and, therefore, can be used according to the invention, eg maleinimide, an iodoacetate, a hydroxymercuribenzoate and Ellman's reagent, among others. Other examples are found in relevant manuals such as those of Voet & Voet (1995) or Lottspeich & Zorbas (1998).

There are also described numerous side chain reactants, which are specific for lysine, such as eg acid anhydrides, (acetic anhydride, N-hydroxy-succinimide, among others, which also find use in the present invention). Lysines have, along with their reactivity, still other advantageous properties for coupling: Due to the electrically charged side chain, it is in most cases next to the protein surface, that is, it is accessible to the solvent , which in biological systems in most cases is water. This accessibility is also necessary for the coupling reagent and should therefore exist.

Free cysteines exposed on the surface, appear in proteins relatively infrequently, in the case of extracellular proteins, these cysteines are integrated in most cases in disulfide bridges, which frequently also stabilize interactions between dimers. However, disulfide bridges can also be cleaved by reduction, such that the contained cysteines are then accessible for modification. If no accessible cysteine residue for a coupling reagent is contained in a polypeptide molecule according to the invention, then it is possible to introduce such residues into a suitable site by mutagenesis. In this case, the knowledge of the spatial structure of the protein is advantageous, since in this way the prediction of favorable amino acid positions for coupling, which are exposed on the surface, is facilitated. However, if multiple cysteine residues are present in a protein, which make site-specific coupling (to a defined cysteine residue, see above) impossible, then these can be removed by site-directed mutagenesis. In this case, the exchange with a serine residue is preferably carried out, which has properties similar to those of the cysteine residue.

However, it is often not possible to obtain suitable amino acid residues for coupling, by mutagenesis in the protein sequence of interest. However, if the protein tolerates insertions of amino acid residues or fusions at the N or C terminus, then peptide sequences, containing the appropriate amino acid residues for coupling, may be deliberately introduced there on the genetic plane.

Therefore, according to another embodiment, the coupling of (I) to (II) is carried out through amino acid residues in a further terminal fusion of peptides to (I).

A check of the accessibility of these terminal peptide fusions can be carried out eg by using a coupling reagent specific for a side chain with chromophore properties, eg the Ellmann reagent in the case of cysteine (also compare Examples). The accessibility of these terminal peptide fusions can be controlled, among others, through their length. An increase in the accessibility of the side chain, which must be coupled, of an amino acid, is possible, for example, by the insertion of so-called spacers, that is to say, of distance elements between the side chain of an amino acid that must be of coupling and protein, in this context in the case of peptide fusions it is a question of additional inert amino acid residues. The residues of glycine and serine are especially suitable for this, since they are only small in size and can thus adopt a very flexible structure.

Depending on the number of functional groups accessible for the coupling reagent in the protein and its specificity, it is possible to differentiate between different types of coupling. For example, in the case of

use of a specific coupling reagent for cysteine only cysteine residues selectively react; but if eg several cysteine residues are present, the exact site of the coupling cannot be predicted, and then one speaks of selective but not directed coupling. If, on the other hand, there is only one cysteine accessible for the coupling reagent, the coupling is carried out in a site-specific manner.

Commercially, a large number of suitable coupling reagents are obtainable (eg from Pierce). A special form of coupling reagents, which can be used in accordance with the invention, is also designated as cross linkers or simply as linkers (Herrmann & Morse, 1973; Takamiya et al., 1975, Reichlin, 1980). .

A linker is defined as a substance, which joins two (or more) molecules by means of a covalent bond. The linkers contain two (or more) reactive (activated) functional groups, the spatial distance of which can be regulated by other chemical groups that link them. The linkers, which have identical functional groups, are designated as homobifunctional, undoing the hetero-bifunctional linkers, which have different functional groups. By means of the appropriate choice of linker substances, it is therefore possible to link together completely different classes of substances.

As an example of a linker that can be used according to the invention, refer to the C-terminal portion of SPC-1-A7-Cys, see Table 2 below.

A special case of protein coupling, however, is also contained without activated linkers: formation of disulfide bridges eg in the case of protein dimerization. Under oxidizing conditions, the sulfhydryl radicals possess a reactivity high enough to form a disulfide, that is, a covalent bond between the two sulfur atoms.

According to the invention, a coupling reaction can proceed either as a single-stage reaction or else in several stages. In the case of a simple dimerization of two identical molecules, which carry only one reactive moiety in each case, it is sufficient to incubate the coupling components with a homo-bifunctional linker, in order to obtain a defined conjugate. In the case of different coupling partners, which also carry different reactive groups, the single-stage reaction is only possible with a corresponding hetero-bifunctional linker.

Alternatively, coupling between different partners in coupling (reactants) with identical reactive groups can, however, be effected in a multi-step process. For this, first only one participant in the reaction is incubated, in most cases with an excess of the linker and the resulting monovalent conjugate is isolated from a reactant and a linker, before it is linked with the second compound reactive through the functional group, which is still free, of the linker. A large number of chemicals, which find use in biochemistry and biotechnology, are commercially available (from the Pierce and Invitrogen entities) in a so-called activated form, that is, they are already bound with a linker which is still reactive.

The degree of coupling, that is to say the relative ratio of the individual components in the conjugate, in the case that several reactive groups are present, is adjustable to a certain extent by means of the stoichiometry of the reactive compounds used. A defined multiple coupling with different partners in the coupling is possible by sequential coupling or by using a different chemical coupling process, eg by coupling the first functional component to cysteines, while functional component 2 engages to the lysines of the corresponding polypeptide. A typical example of this is the coupling of pairs of fluorescence donors and acceptors for measurements by FRET (acronym for "Fluorescence Resonance Energy Transfer" = energy transfer by fluorescence resonance). Suitable combinations of dye molecules are available from Invitrogen.

Regardless of the chosen coupling mechanism, leading to the conjugate according to the invention, mention must again be made of the fact that the ability of the present conjugates to function was not possible in this way and that it was surprising. With regard to this, reference is also made to the preceding explanations.

In addition to this, it should be mentioned that for a person skilled in the art, it was not expected that conjugates such as those disclosed in the present invention could be produced, remaining conserved or even increasing the full functionality of the individual components.

The size relationships between the polypeptide molecules according to the invention, on the one hand, and the functional components, on the other hand, are very diverse. Partially, the molecules that must be attached, for example a phycoerythrin, are 10-12 times larger than polypeptide molecules, but partially, such as the Oyster fluorescent dye, they are also smaller. Surprisingly, especially in the

In the case of bound molecules that are much larger, both the structure of the polypeptide molecule and the binding affinity to the ligand remain preserved. This fact was not to be expected in this way.

As already outlined above, the side chains for coupling to the functional component are located outside the binding surface of (I) with the ligand, so as not to disturb the functionality of the binding to the ligand.

In accordance with another preferred embodiment - as mentioned above - the terminal fusion of peptides a (I) contains one or more cysteine residues or one or more lysine residues, these amino acid residues preferably not participating in the interaction of (I) with the ligand.

The functional component (II) is chosen from the set consisting of polypeptides and proteins, organic polymers, sugars, low molecular weight substances, peptides as well as derivatives of these substances. For the principles of binding and coupling reagents, see the explanations above.

According to a preferred embodiment, the functional component (II) is a peptide, a polypeptide or a protein, preferably a chromophore protein, an enzyme, an immunoglobulin, an immunoglobulin derivative, a toxin or a polypeptide of according to I.

If the functional component (II) is a polymer, it is preferably chosen from dextran, a polymethacrylate, a Sepharose, an agarose, a vinyl polymer, a polystyrene, a silica gel, a cellulose or a poly (ethylene glycol), or a derivative of a polymer.

In case the functional component (II) is a low molecular weight substance, it is preferably a dye, biotin, digoxigenin, a heavy metal, a chelate-forming compound, a radioisotope, an antibiotic or a cytotoxic substance. .

The ligand that specifically binds to component (I) is chosen from the set consisting of polypeptides, peptides and proteins.

In the case that in this context it is a polypeptide or a protein, immunoglobulins and derivatives of immunoglobulins, proteins obtained from a blood plasma, factors and agents that inhibit blood coagulation are preferably used, growth factors, interleukins, cytokines, receptor proteins, virus proteins and cell surface markers, preferably CD14, CD25 and CD34.

In the event that the ligand is a peptide, it will preferably be an affinity tag, preferably the S-brand, the T7-brand, the His-brand, the Strep brand, the Myc-brand or the FLAG-brand, or a viral peptide.

According to a preferred embodiment, component (II) of the conjugate according to the invention consists of one or more ubiquitin-based polypeptides, which are identical to (I), and which are linked by covalent bonds with it, thereby which an increase in the affinity to the ligand of (I) is achieved through avidity effects. For a detailed explanation of this refer to the explanations provided above.

Furthermore, component (II) is preferably a polypeptide, a protein or a polymer, which has been covalently linked multiple times to component (I), thereby achieving increased affinity for the ligand of ( I) through avidity effects. Alternatively, component (I) is a polypeptide or polymer, which after covalent bonding with component (I) becomes part of covalent or non-covalent bonding with other conjugates of this type, thereby which an increase in affinity for the ligand of (I) is achieved through avidity effects.

In accordance with a preferred embodiment, component (I) is one of the SPU11-3-A1 molecules (SEQ ID NO: 12 and 13).

The invention describes not only the exact nucleic acid sequences, but also variants thereof. According to the invention, "variants" are in particular those nucleic acids, in which one or more substitutions, insertions and / or deletions occur in comparison with the nucleic acids defined in SEQ ID NO. In the case of these, preferably at least 1, but also 2, 3, 4 or more nucleotides are missing next to one or both ends of the nucleic acids, or also inside the nucleic acids, or they are replaced by other nucleotides.

The nucleic acids of the present invention therefore encompass also nucleic acids, having sequences, that are essentially equivalent to the nucleic acids of the respective SEQ ID NO. Nucleic acids according to the invention can eg have at least about 80%, typically at least about 90% or 95% sequence identity with the nucleic acids of SEQ ID NO.

The concept of "nucleic acid sequence" refers to a nucleotide heteropolymer or the sequence of these nucleotides. The concept of "nucleic acid" as used in this context encompasses both an RNA, a DNA, including a cDNA, a genomic and synthetic DNA (or respectively synthesized by chemical means) as well as bases bound to other polymers , such as ANP.

The invention also relates to variants, which hybridize with the nucleic acids according to the invention under moderately stringent conditions.

By the concept of "stringent hybridization and wash conditions" is generally meant reaction conditions, under which only duplex molecules are formed between the oligonucleotides and the desired target molecules (perfect hybrids) or respectively only Detects the desired target organism. In this case, the term "stringent hybridization conditions" is understood in particular to mean 0.2 x SSC (0.03M NaCl, 0.003M sodium citrate, pH 7) at 65 ° C. In the case of shorter fragments, for example oligonucleotides based on up to 20 nucleotides, the hybridization temperature is below 65 ° C, for example, above 55 ° C, preferably above 60 ° C, but in any case below 65 ° C. Stringent hybridization temperatures are dependent on the size or respectively the length of the nucleic acid and its nucleotide compositions, and must be determined by one skilled in the art by manual assays. Moderately rigorous conditions are achieved for example at 42 ° C and with a wash in 0.2 x SSC / 0.1% SDS at 42 ° C.

The respective temperature conditions can be diverse depending on the chosen test conditions and depending on the nucleic acid sample, which must be investigated, and then have to be adapted accordingly. Detection of the hybridization product can be carried out, for example, by autoradiography in the case of radioactively labeled molecules, or by fluorimetry in the case of the use of fluorescently labeled molecules.

A person skilled in the art can adapt the conditions in a manner known per se to the chosen investigative procedure, in order to actually achieve moderately rigorous conditions and to enable a specific detection procedure. Suitable stringent conditions can be determined, for example, with the aid of reference hybridizations. A suitable concentration of nucleic acids or respectively oligonucleotides must be used. Hybridization must take place at a suitable temperature (the higher the temperature, the weaker the hybrid bonds will be).

According to an embodiment, the present invention relates to a process for the preparation of a conjugate as defined above, starting from component (I) with a known sequence, which comprises the following steps:

-

Identification for coupling of suitable amino acid residues by analysis of the spatial structure of the protein, preferably of residues located outside the interaction surface of (I) with the ligand;

-

Activation of a partner in the coupling by means of a suitable coupling reagent;

-

Carrying out the coupling reaction:

-

Isolation of the conjugate; and

-

Detection of the functionality of both components of the conjugate.

A modified procedure for the preparation of a conjugate of the invention comprises, starting from component (I) with a known sequence, in which no suitable amino acid residue for coupling had been identified, the following steps:

-

Introduction of suitable amino acid residues for coupling by substitution, insertion or fusion, preferably of residues exposed on the surface outside the interaction surface of

(I) with the ligand;

-

Detection of the accessibility of the introduced amino acid residues;

-

Detection of the functionality of component (I) modified in this way;

-

Activation of a partner in the coupling by means of a suitable coupling reagent;

-

Carrying out the coupling reaction;

-

Isolation of the conjugate; and

-

Detection of the functionality of the two components of the conjugate.

More detailed explanations regarding coupling procedures, etc., are found above.

In this context a pharmaceutical composition is described, encompassing a conjugate produced according to the invention and a pharmaceutically compatible vehicle.

In the pharmaceutical composition, the conjugate is mixed with suitable vehicles or suitable vehicle substances in certain doses, so that the disease is treated or at least alleviated. Such a composition may contain (in addition to the active substances and the vehicle) dilution agents, fillers, salts, buffers, stabilizing agents, solubilizing agents and other materials, which are well known in the art. technique. The concept of "pharmaceutically compatible" defines a non-toxic material, which does not disturb the efficacy of the biological activity of the active constituent substance or respectively of the active substance. The choice of vehicle depends on the route of administration.

The pharmaceutical composition may additionally contain other agents, which increase the activity of the active substance or that complement the activity or its use in the case of treatment. Such additional factors and / or agents may be contained in the pharmaceutical composition, in order to achieve a synergistic effect or to minimize side effects or respectively undesired effects.

Techniques for the formulation or respectively preparation and administration of the conjugates herein application can be found in "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA, Last edition. A therapeutically effective dose further refers to an amount of the compound, which is enough to achieve an improvement in symptoms, for example, a treatment, a cure, a prevention or improvement of such states. Suitable routes of administration may include, for example, one oral, rectal, transmucosal or intestinal administration, and one parenteral administration, inclusive intramuscular, subcutaneous, intramedullary injections, as well as intrathecal injections, directly intraventricular, intravenous, intraperitoneal or intranasal. Intravenous administration to a patient is preferred.

A defined conjugate such as in this context can be used in diagnosis, therapy and affinity chromatography.

The exposed coupling methods and the data obtained with them make possible the most diverse uses of the conjugates, among them the use of the conjugates in activity chromatography. Examples of these are the use of protein A for the purification of antibodies and the purification of proteins from blood plasma, growth factors or influenza vaccines, by means of affinity chromatography as well as the purification of proteins with appendage appendages, produced by recombinant means, or the depletion of endotoxins or respectively albumins. By means of a coupling with matrices, it is also conceivable the use in blood plasmaphoresis or bioremediation.

Another sector of use is established in the diagnosis. In this case, jobs in blood bank screening for bacterial or viral infections or in classical detection techniques such as an ELISA or new developments such as in a Luminex system are conceivable. Also in diagnosis but also in therapy, these conjugates can be used in cell separation.

The use of the conjugates prepared according to the invention in therapy is also possible, in particular the general use as transport molecules. Other uses in therapy could be found in gene therapy, in the case of deliberate targeting by a polypeptide molecule according to the invention and coupling to systems for gene transfer. Also, through deliberate targeting and coupling to a bacterial toxin, use as an immunotoxin in therapy would be established.

The present invention will be described hereafter with the aid of various figures and accompanying examples, which however should not restrict the scope of the invention, but only illustrate it.

Figure 1: Spatial structure of crystalline gamma II. The de novo generated binding surface in the N-terminal domain has been shown in yellow. The location at the C terminus of the lysine residues located outside the fixation surface has been highlighted by spherical red caps.

Figure 2: Sensograms of Biacore experiments for competition in binding AffilinTM variants SPC1-A1 (A), SCP1-A7 (B) and SPC1-G3 (C) to a CM5 chip with an IgG-Fc immobilized. For the experiments 180 RU of a polyclonal IgG-Fc were immobilized. For binding competition, the indicated concentrations of the variants and IgG-Fc were used. HBS-EP with a circulation rate of 30 µl / min was used as the elution buffer.

Figure 3: Concentration-dependent ELISA for detection of SCP7-E9 binding to proNGF. The microtiter plate was coated with 10 µg / ml proNGF. Served as the detection antibody was a conjugate of a human crystalline anti-gamma II antibody and a POD in a dilution of 1: 1,000. The absorption values shown are average values of two parallel measurements. An apparent KD value of 200 nM could be calculated.

Figure 4: Concentration-dependent ELISA for detection of binding of SCP1-A7-Cys to human IgG-Fc. The microtiter plate was coated with 10 µg / ml of the IgG-Fc. Served as the detection antibody was a conjugate of a human crystalline anti-gamma II antibody and a POD in a dilution of 1: 1,000. The absorption values shown are the average values of two parallel measurements. An apparent KD value of 233 nM could be calculated.

Figure 5: Sensograms of the experiments according to Biacore about the binding of a SCP1-A7BB-PE conjugate to a CM5 chip with an immobilized IgG-Fc. 3,000 RU of the IgG-Fc were immobilized and concentrations of 121 nM (in red color), 75 nM (in green color) and 6 nM (in blue color) of a SPC1-A7BB-PE conjugate were conducted through the chip . The association phase was 1 min, followed by a dissociation phase for 3 min. HBS-EP with a circulation rate of 30 µl / min served as the elution buffer. From the curves, a macroscopic KD value of 15 nM could be calculated.

Figure 6: Sensograms of the experiments according to Biacore to check the binding of SPC1-A7 Oyster556 to a CM5 chip with an immobilized IgG-Fc. 1,000 RU of IgG-Fc was immobilized on the chip and SPC1-A7 Oyster556 was driven in concentrations of 1 µM (blue color) and 5 µM (red color) through the chip. The association and dissociation phases were in each case 3 min. HBS-EP with a circulation rate of 30 µl / min served as the elution buffer.

Figure 7: Detection of binding of a conjugate of an AffilinTM and a POD to an IgG by an ELISA. 10 µg / ml of a human IgG was immobilized to a microtiter plate. Various dilutions of the Affilin ™ and POD conjugate were incubated in a PBS for 1 hr on the microtiter plate. After a washing step, the activity of the bound POD was detected by means of a TMB substrate solution.

Figure 8: Sensograms of the experiments according to Biacore about the binding of SPU11-3-A1_Cys to NGF. 200 RUs of SPU11-3-A1_Cys were coupled to the CM5 chip with the aid of PDEA and various concentrations of NGF were driven through the chip. PBS (1mM EDTA, 0.005% Surfactant P20) with a circulation rate of 30 µl / min served as the elution buffer. A KD value of 46 nM could be calculated from the curves.

Figure 9: Sensograms of the experiments according to Biacore about the binding of SPC7-E9 to a CM5 chip with proNGF. 280 RU of proNGF were immobilized and various concentrations of proNGF were driven through the chip. HBS-EP with a circulation rate of 30 µl / min served as the elution buffer. A KD value of 1.4 nM could be calculated from the curves.

Figure 10: Elution of proNGF from an affinity column with SPC7-EP9. 400 µg of purified proNGF (moment 0, pink line drawn with dashed lines) was applied and then rinsed with 20 column volumes of the elution buffer. Elution was carried out with 0.1M glycine pH 2.2 (green line). Elution was carried out at a flow rate of 1 ml / min. Protein detection was carried out at 280 nm (blue line).

Figure 11: SDS-PAGE of the separation of proNGF from a BSA solution and from a crude extract of E. coli (vlnr) lane 1: marker proteins, lane 2: BSA standard, lane 3: proNGF standard, lane 4: mixture of BSA and proNGF standards (application), lane 5: material that has passed through the column, lane 6: elution with 0.2 M glycine (pH 2.2), lane 7: empty, lane 8 : mixture based on a crude extract of E. coli (BI 21) and a proNGF standard, lane 9: material that has passed through the column, lane 10: elution with 0.2 M glycine (pH 2.2 ).

Example 1

Selection of the AffilinTM variant that binds IgG-Fc and proNGF from the human crystalline gamma II library, expression and purification

Starting from the CR20 human crystalline gamma library, a selection process was carried out with the help of the phage display system. In this case, after one round several variants of Affilin ™ could be selected and isolated, which in the individual phage ELISA showed a specific binding to an IgG-Fc. It should be mentioned that the concept of "Affilin ™", used here, corresponds to the component of the polypeptide molecule according to the invention of the conjugate, which is based on ubiquitin or crystalline gamma, and which has a property compared to the wild type modified binding for specific binding to a ligand. After a cloning change of the genes in the expression vector pET20b (from Novagen) Affilin ™ variants were overexpressed by recombinant means in E. coli (BL21 (DE3), from Stratagene), and then purified in two stages of chromatography (IMAC and filtration through a gel). In a concentration-dependent ELISA and in experiments according to BIACORE, a specific binding to IgG-Fc could be determined with a dissociation constant located in the nM region.

For the selection of AffilinTM variants that bind to an IgG-Fc, 1 ml of the CR20 library (6.5 x 1010 cfu (colony forming units)) was incubated in 1 medium of 12 x YT with glucose to 2% and 100 µg / ml ampicillin at 37 ° C and 220 rpm until an optical density of DO600 = 0.4 is reached. The bacterial culture was then incubated with a 10-fold excess of M13K07 helper phages (from Invitrogen, Karlsruhe, Germany) for infection for 1 hr at 37 ° C and 100 rpm. The bacterial suspension was then centrifuged for 20 min at 1,000 x g, and the pellet was resuspended in 112 x YT medium with 8mM GSH, 100 µg / ml ampicillin, and 50 µg / ml kanamycin. Phage production or phage release respectively was carried out at 30 ° C and 200 rpm overnight. For the isolation of phages, the protocol described by Kay, Winter & McCafferty (1996) was used.

1 ml of the isolated phages (1.4 x 1014 cfu) was blocked with 1 ml of 6% BSA in a PBS for 1 hr at room temperature (RT). Meanwhile, 10 wells of a microtiter plate (NUNC), which had been coated overnight at RT, were coated with 100 µl of a 10 µg / ml solution of monoclonal IgG-Fc (from Roche) in PBS, and washed three times with PBS; Tween 20 0.1%. The free binding sites of the wells were then blocked with 300 µl of a PBS (3% BSA, 0.5% Tween 20) in each case for 2 h at RT. This was followed by repeated washing of the wells three times with PBS (0.1% Tween 20). After an addition of in each case 100 µl of the phages blocked by each well, an incubation was carried out for 1 h at RT and 20 rpm. Phages not fixed or weakly fixed respectively were removed by a repeat wash 2 times with 2 x PBS, a repeat wash twice with 2 x PBS, 3% BSA and finally repeated 2 times with 2 x PBS. Phages still fixed were eluted by adding 100 µl / well of 100mM triethylamine and incubating for 10 min at RT. To neutralize the phages eluted in a basic medium (in total 1 ml), they were mixed with 500 ml of 1M Tris / HCl, pH 7.4. After this, the wells were washed three times with PBS.

The strongly fixed phages, which had remained in the microtiter plate despite elution with triethylamine, were incubated directly for reinfection with 100 µl of an exponentially growing cell culture (OD600 = 0.4) of XL1-Blue for 30 min at 37 ° C. For the reinfection of XL1-Blue cells with the phages eluted in a basic medium, 750 μl of the neutralized eluted material were incubated with 9 ml of XL-1-Blue cells with an OD600 = 0.4 for 30 min at 37 ° C. The phage reinfected cells eluted in basic medium and strongly fixed phage were then pooled, plated in 16 x 16 cm dishes with SOBAG medium (including ampicillin) and incubated overnight at 37 ° C. . After a coating process, approximately 2,000 clones were obtained, which were separated by flooding the plates with approximately 12.5 ml of a 2 x YT medium; 20% glycerol and stored at -80 ° C.

For the cultivation of the individual phages, the cell pool obtained after the first round of overcoating was plated onto a selection medium (SOBAG) and incubated overnight at 37 ° C. From the SOBAG plate 92 individual clones were transferred to 24 x 5 ml deep well plates with in each case 2 ml / well of 2 x YT medium with 2% glucose and 100 μg / ml Amp and incubated overnight at 37 ° C and 180 rpm. Additionally, for each plate an individual colony (X11-blue) with the gene for the wild-type human crystalline gamma was driven concomitantly in the phagemid vector as a control. Sterile, 24 x 5 ml deep well plates were inoculated with each case 2.5 ml / well of 2 x YT medium with 2% glucose and 100 µg / ml Amp with a 1% inoculum of the Overnight culture, and the bacterial cultures were incubated at 37 ° C and 180 rpm to an OD600 of approximately 0.4. The cultures were then infected with 2.5 µl per well of M13K07 helper phages with 1013 cfu / ml and incubated for 1 hr at 37 ° C and 100 rpm. The bacteria were then pelleted by centrifugation, the supernatant was discarded, and the pellets were resuspended in 2.5 ml per well of 2 x YT medium, 8 mM GSH, 100 µg / ml ampicillin, 50 µg / ml of Kanamycin and incubated overnight at 30 ° C and 200 rpm. To obtain the supernatant material from the phages, the plates were centrifuged at 4,600 rpm. Phage precipitation and sedimentation were carried out as described by Kay, Winter & McCafferty, and the phage pellet was resuspended in approximately 200 µl of a PBS, 3% BSA, pH 7.4 . By this procedure, the phages could be concentrated and then used in an ELISA.

For this, the wells of a NUNC plate were coated with 100 µl of an antigen solution (10 µg / ml of a human monoclonal IgG-Fc, or respectively BSA) overnight at 4 ° C. The ELISA plate was incubated the following day with a blocking buffer (one PBS, 3% BSA, 0.1% Tween 20, pH 7.4) for 2 h at room temperature. After the wells had been washed with a wash buffer (a PBS, 0.1% Tween 20, pH 7.4), 100 µl of the phage preparations were each introduced into the wells and incubated for 1 has the TA. After renewed washing of the wells with a washing buffer (a PBS, 0.1% Tween 20, pH 7.4), the anti-M13 monoclonal antibody (conjugated to a POD; from MoBiTec, Göttingen) was applied in a dilution of 1: 5,000 in a PBS, pH 7.4 (100 µl / well) and incubated again for 1 h at RT. After this, the wells were washed 3x (times) with a wash buffer (a PBS, 0.1% Tween 20, pH 7.4) and 3x with a PBS, and the color reaction was started with TMB Plus (from Kementec, Denmark) (100 µl / well). After 20 minutes, the color reaction was interrupted by an addition of 0.2M H2SO4. The yellow coloration obtained was recorded by reading at 450 nm (reference wavelength: 620 nm) and documented.

From phage preparations, which showed a clear signal for binding to monoclonal IgG-Fc and barely detectable binding to BSA, the genes for crystalline gamma II variants were

sequenced with the pCAN700 primer. Three resulting clones SPC1-A1, SPC1-A7 and SPC1-G3 were cloned into the pET20b expression vector through the restriction cleavage sites with NcoI and BstEII and introduced into the BL21 expression strain. (DE3), pUBS520 (from Stratagene).

Cells were grown in 2 x YT medium with 100 µg / ml ampicillin and 50 µg / ml kanamycin until an optical density of DO600 = 0.6 - 0.8 at 37 ° C and 200 rpm, and then expression of the recombinant protein was induced with IPTG (final concentration 1mM). After growth for four hours at 30 ° C and 200 rpm, cells were harvested by centrifugation (at 6,000 x g, 20 min, 4 ° C). Disintegration was carried out in an NPI-10 buffer (from Qiagen) by means of lysozyme (0.1 mg / ml) and ultrasound (6 X 15 s, mediating cooling with ice) in the presence of 5 mM beta-mercaptoethanol. After centrifugation (at 40,000 xg, 30 min, 4 ° C) the supernatant was applied to a column with IMAC (HiTrap Chelating HP, from Amersham Bioscience) and washed with NPI-20 (Qiagen, + beta-mercaptoethanol 5 mM) (20 column volumes). Elution was performed using a linear gradient with NPI-500 (Qiagen, + 5mM beta-mercaptoethanol) in 30 column volumes. The fractions, containing crystalline gamma II, were analyzed by SDS-PAGE, corresponding samples were pooled and applied on a filtration column through a gel (1.6 x 60, Sephadex 75, from Amersham Biosciences). PBS served as the elution buffer, with a circulation rate of 0.75 ml / min. Analysis of the filtration through a gel was carried out by SDS-PAGE, the fractions containing crystalline gamma II were pooled and stored at 4 ° C. Affilin ™ variants had a purity of> 95% after this purification process (SDS-PAGE).

The binding properties of the purified proteins were then tested as described above in a concentration dependent ELISA. In this case, different concentrations (100 nM - 10 µM) of the Affilin ™ variants were used and a crystalline anti-gamma II antibody (a monoclonal antibody conjugate with POD) was used as the detection antibody. In this case, it was shown that all three Affilin ™ variants tested possessed specific binding against human IgG-Fc and that nonspecific binding to BSA or microtiter plate was not detectable. Concomitantly run wild-type human crystalline gamma II showed no binding to IgG-Fc, BSA or microtiter plate.

In some experiments according to BIACORE, the dissociation constants of the three Affilin ™ variants were determined. For this, approximately 180 RU of a human IgG-Fc (50 µg / ml in 50 mM Na citrate, pH 5.0) were immobilized on a CM5 chip. The free binding sites were finally deactivated by 1M ethanolamine (pH 8.5).

In the case of a flow rate of 30 μl / min, 6 different concentrations (166 nM - 1μM) were subsequently conducted for 180 s through the chip. The chip was then rinsed with HBS, 0.005% Surfactant P20 (from Biacore) at the same flow rate for 180 s. From the resulting sensorgrams, with the help of the BiaEvaluation software (from Biacore, Uppsala, Sweden) the following dissociation constants of the AffilinTM variants against an IgG-Fc are determined: SPC1-A1 with 230 nM, SPC1-A7 with 280 nM and SPC1-G3 with 800 nM. In some competition experiments, the specific binding of Affilin ™ variants to an IgG-Fc and not to the chip matrix could be detected (Fig. 2).

Analogously to the selection of the SPC1-A1, SPC1-A7 and SPC1-G3 variants, the Affilin ™ SPC7-E9 variant was isolated against the target molecule proNGF. The dissociation constant could be determined using BIACORE measurements with 1-6 nM (Fig. 3).

Example 2

Selection of AffilinTM variants from the human ubiquitin library (UB10) against a cysteine knot protein - expression and purification

Provision of an artificial ubiquitin gene for the selection of modified proteins with a binding affinity generated from new

The genetic technology works were carried out, as long as nothing else had been indicated, according to the usual classic protocols for an expert in the specialty such as, for example, Sambrook et al. (1989).

For the production of the DNA sequence (SEQ ID NO: 25) for a modified ubiquitin protein framework with the substitutions Ile44Ala, Lys48Arg, Arg54Leu, Val70Ala, Arg72Leu, Gly75Ala, as well as with the Gly76 deletion, as a starting point for the Obtaining artificial binding proteins was carried out as follows: For the synthesis of the gene, a PCR reaction was carried out in a volume of 50 µl, in which 2.5 µl of the six were present as templates in each case. oligodeoxynucleotides (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID NO: 29, SEQ ID NO: 30, SEQ ID NO: 31; in each case 0.1 μM), representing in their order of succession of the base pairs in total to the gene to be synthesized. The sequences of the oligodeoxynucleotides used corresponded in each case to segments with a length of 40 to 50

base pairs of the DNA strand encoding or respectively non-encoding the artificial gene, and alternately overlapping their 3 'and 5' ends with approximately 15 bases. Additionally, the batch each contained 2.5 µl of flanking primers ((SEQ ID NO: 32, SEQ ID NO: 33; 10 µM) as well as 5 µl of a 10 x Taq buffer (100 mM Tris / HCl, pH 9.0, 500 mM KCl, 1% Triton X-100 (v / v)), 3 µl of 25 mM MgCl2 and 4 µl of a mixture of dNTP's (dATP, dCTP, dGTP, dTTP in each case 2 , 5 mM) After filling with H2O the reaction batch was heated in the thermocycler in order to carry out denaturation for 2 min at 94 ° C. Then 2.5 U of Taq polymerase were hot added ( Promega) (Hot Start) and the PCR program was started. In 25 cycles, each case was incubated for 1 min at 94 ° C, for 1 min at 55 ° C and for 1.5 min at 72 ° C. A final incubation was carried out for 5 min at 72 ° C.

The desired PCR product was identified by analytical electrophoresis through an agarose gel and purified from the batch with the aid of the MinElute Reaction Cleanup-Kit (from Qiagen). 1.0 ng of the isolated DNA was used as a template for a second amplification, which this time was done using Pfu polymerase (from Promega) also in a volume of 50 µl. For this, 5 µl of the concomitantly supplied 10 x Pfu buffer (200 mM Tris / HCl, pH 8.8, 20 mM MgCl2, 100 mM KCl, (NH4) 2SO4 100 mM, 1% Triton X-100 (v / v), 1 mg / ml BSA) as well as 4 µl of a mixture of dNTP's and filled with H2O. Additionally, the batch contained flanking primers (SEQ ID NO: 32, SEQ ID NO: 33; 10 µM) for the introduction of suitable cutting sites. The desired PCR product was isolated by preparative electrophoresis through an agarose gel and inserted using the Zero Blunt® TOPO® PCR Cloning Kit (from Invitrogen) according to the manufacturer's data in the pCR® cloning vector. 4Blunt-TOPO®. Concomitantly supplied chemically competent cells were transformed with the corresponding ligation reaction batch and plated on an agar plate with LB / Amp / Kan medium. The plate was incubated for 16 hours at 37 ° C, and the colonies that had grown were analyzed for the desired ligation product. For this, a plasmid DNA was prepared on the mini-scale using the Qiagen entity plasmid isolation kit according to the manufacturer's data and subjected to restriction digestion with the NdeI and XhoI DNA endonucleases (from New England Biolabs), whose recognition sequences had been introduced by the flanking primers into the PCR product. With plasmids, which had the expected cut pattern, an analysis of the DNA sequence in the region of the inserted gene cassette was carried out with the help of the Taq DNA polymerase. In this case, the CycleReader ™ AutoDNA Sequencing Kit (from Fermentas) according to the manufacturer's data was used, as well as 0.5 µg of a plasmid DNA and 1.0 pmol of the corresponding fluorescence-labeled primer. The newly synthesized DNA strand in this case was marked during the polymerase reaction and was statistically terminated, but in a base-specific manner, by incorporation of di-deoxynucleotides. The resulting fluorescent DNA fragments were then separated on a Liquor sequencing automat (treatment liquid) by electrophoresis through a polyacrylamide and urea gel and became visible as a banding model for A, C, G and T in contiguous traces.

Gene cassettes with the correct DNA sequence were cut by preparative restriction digestion with NdeI / XhoI from the pCR®4Blunt-TOPO® cloning vector and isolated by preparative electrophoresis through an agarose gel. The insertion of the gene for the modified ubiquitin protein framework was carried out in the expression vector pET20B (-) (from Novagen) for the production of the corresponding protein or respectively in the fasmid vector pMUBI-1 for the construction of a library of ubiquitin variants.

Production of a library of ubiquitin variants

For consecutive 8-codon site-directed mutagenesis at the amino or carboxy terminus of the synthetic ubiquitin gene, two consecutive PCR reactions were performed. The first stage of amplification took place using Pfu polymerase (from Promega) in a volume of 10 x 50 µl. For this, 5 µl of the 10 x Pfu buffer supplied concomitantly as well as 4 µl of a mixture of dNTP's were used for each batch and filled with H2O. In addition, the batch each contained 2.5 µl of flanking primers (SEQ ID NO: 34, SEQ ID NO: 35; 10 µM) for introduction of the desired base pair exchanges. 1.0 ng of pMUBI-1 was used as templates, carrying the synthetic unmutated gene of ubiquitin. After an addition of 2.5 U of Pfu polymerase (see above) it was incubated in 25 cycles during each case 1 min at 94 ° C, 1 min at 60 ° C and 1.5 min at 72 ° C . A final incubation was carried out for 5 min at 72 ° C. For the selective degradation of the matrix DNA used, 10 U of DpnI were added for each reaction batch and incubated for 1 hour at 37 ° C. The desired PCR product was isolated by preparative electrophoresis through an agarose gel and from the QIAquick Gel Extraction Kit (from Qiagen).

The second stage of amplification was carried out in a batch of 1,000 µl, using approximately 1.0 ng of the product obtained in the first stage of PCR and Taq polymerase. The reaction batch - adapted to a 20-fold volume - was pipetted as above from a 10 x Taq buffer, with 25mM MgCl2, a mixture of dNTP's as well as the flanking primers (SEQ ID NO: 36, SEQ ID NO: 37; 10 µM), which had been biotinylated near their 5 'ends and which carried in each case recognition sequences not compatible with each other for the endonuclease SfiI. After filling with H2O, 2.5 U of the

Hot Taq polymerase (see above) and the PCR program started. In 25 cycles, 1 min at 94 ° C, 1 min at 60 ° C and 1.5 min at 72 ° C was incubated in each case. A final incubation was carried out for 5 min at 72 ° C.

The subsequent dissociation of the obtained amplification product was carried out directly in the PCR reaction batch. For this, in a total volume of 4,000 μl, the complete solution of the PCR reaction was mixed with the corresponding volume of buffer 10 x II (100 mM Tris / HCl, pH 7.9, MgCl2 100, 500 mM NaCl, dithiothreitol 10 mM) supplied concomitantly, a solution of 10 x BSA and H2O. In addition 4,000 U of the restriction enzyme SfiI (from New England Biolabs) were added and incubated for 16 hours at 50 ° C. DNA was isolated from the batch with the aid of the MinElute Reaction Cleanup Kit (from Qiagen) and collected in 400 µl of sterile H2O. For separation of the PCR product not cut with SfiI, the isolated DNA was mixed with the same volume of a "Binding-Solution" (from Dynal), containing 1.0 mg / ml of magnetic beads with streptavidin coupled to the surface ("Dynabeads Kilobase Binder"), and incubated for 4.5 hours in a roller mixer at room temperature (RT). The beads were precipitated with the DNA, which was eventually still present, while the DNA completely cut with SfiI, which should no longer have any biotinylated ends, remained in the supernatant and was precipitated overnight. The ubiquitin gene cleaved with SfiI and mutagenized in the desired positions obtained in this way, was dissolved in sterile H2O, desalinated again using the QIAquick PCR Purification Kit (from Qiagen) and finally had a concentration of 200 fmol / μl in H2O.

For the previous preparation of the receptor vector, the pMUBI-1 plasmid was cut with SfiI according to the manufacturer's data and the largest fragment (of the vector) was isolated by preparative electrophoresis through an agarose gel and with the QIAquick PCR Extraction Kit (from Qiagen). In order to prevent intramolecular ligation, its 5 'ends were dephosphorylated. For this, 0.5 U of the alkaline phosphatase from shrimp (Pandalus borealis) as well as the concomitantly supplied buffer in a total volume of 200 μl were used. The mixture was incubated for 90 min at 37 ° C, the DNA was isolated from the batch with the help of the QIAquick PCR Purification Kit (from Qiagen) and was desalinated again (with the QIAquick PCR Purification Kit). The DNA of the vector fragment finally had a concentration of 50 fmol / µl in H2O).

For ligation, 1.6 pmoles of the PCR fragment and 8.0 pmoles of the pMUBI-1 vector fragment were incubated in the presence of 2 U of T4 DNA ligase (from GibcoBRL) in a total volume of 1,600 µl (Tris / 50mM HCl, pH 7.6, 10mM MgCl2, 1mM ATP, 1mM DTT, 5% (w / v) PEG-8,000) for three days at 16 ° C. After heating the batch to 65 ° C, the DNA was precipitated for 15 min. For this, in each case 100 µl of the reaction solution were mixed with 100 µl of ethanol as well as 10 µl of 5M NaAc, pH 3.0, and stored for 16 hours at -20 ° C. Then, it was centrifuged (for 60 min, at 12,500 g), washed with ethanol (70% v / v, at -20 ° C), it was centrifuged again and the precipitated DNA was finally dissolved in 60 µl of H2O sterile.

For electroporation, the Gene Pulser® II system (from Biorad) was used, as well as cuvettes with a distance between the electrodes of 1.0 mm (from Biozym) at 4 ° C in the cold room. In each case 3.5 µl of the solution obtained above, electrocompetent E. coli XL1Blue cells (from Stratagene) were transformed according to the manufacturer's data. The obtained cell suspension was plated on five agar plates (20 x 20 cm) with LB and chloramphenicol medium. Plates were incubated for 16 hours at 37 ° C and colonies that had grown were counted. The constructed library accordingly contained 2.8 x 107 independent clones, each of which should be present 10,000 times in the library. The colonies were then washed by drowning in 100 ml total SOC medium with 10% glycerol (v / v = volume / volume) and stored in 1.0 ml aliquots at -80 ° C. From the clones obtained, with the aid of the DNA-Miniprep Kit from Qiagen entity, 12 arbitrarily selected fasmid vectors were isolated and the DNA sequence in the region of the mutagenized ubiquitin gene was analyzed. In this case, all the clones had functional sequences.

-

 that is, no displacement of the reading frame by insertions or deletions - as well as totally different qualitative substitutions together with the mutagenized positions. No random exchange occurred outside the mutagenized regions.

On the basis of this library, based on human ubiquitin, in a manner analogous to Example 1, by the method known to a person skilled in the art of the phage display system, a selection was carried out. Smaller deviations resulted only in relation to the selection of the antibiotic used (chloramphenicol instead of ampicillin).

A growth factor of the cysteine knot protein family served as the target. Affilin ™ ubiquitin SPU11-3-A1 had a dissociation constant, determined in the ELISA, located in the nM region, and was used for coupling studies in the BIACORE system.

One of the variants selected from these, SPU11-3-A1, was introduced by cloning into the expression vector pET20b through the restriction cleavage sites with NdeI and XhoI. The culture conditions and the purification procedure were identical (o) to those (al) of the SPC AffilinTM (IMAC, gel filtration), as explained in Example 1. For the detection of the properties of Fixation A concentration dependent ELISA was carried out. In this case, different concentrations (10 nM - 1 µM) of the AffilinTM variant were applied on the microtiter plate (MTP) coated with the target molecule and a

anti-ubiquitin polyclonal antiserum (from Sigma) as a primary detection reagent. After incubating (for 1 h) at RT, the MTP wells were washed 3x (times) with PBS, and in a second step a monoclonal antibody conjugate (anti IgG, from Sigma) was used with a POD as the detection antibody. In this case, it was revealed that the Affilin ™ variant tested possesses a specific binding against human NGF, and that a nonspecific binding to BSA or microtiter plate was detectable. Concomitantly conducted wild-type human ubiquitin did not show any binding to NGF, BSA, or the microtiter plate.

Example 3

Fusion at the C-terminus of AffilinTM with a cysteine-containing peptide linker, for selective coupling to different molecules

The following Example shows that the AffilinTM SPC1-A7 variant, which binds to an IgG-Fc, could be selectively coupled to various molecules through a cysteine located at the C-terminus.

The variant AffilinTM SPC1-A7 used has, together with the 7 cysteines located inside the protein, in variable position 4 and a cysteine accessible for the solvent and therefore free. This was first replaced by a serine using QuickChange® PCR. Starting from this modified Affilin ™ (SPC1-A7BB) at the C terminus, two glycins and one cysteine were inserted as well as, in addition to the six histidines, four other histidines were present by means of a QuickChange® PCR. The prolonged affinity mark on 10 histidines should serve for enhanced purification. In the Ellmann reagent titration experiments, the introduced cysteine was shown to be unsuitable for coupling experiments due to shuffling events of cysteines with other cysteines present in the Affilin ™ variant. Because of this, the cysteine was replaced at the DNA level by a serine (TCT), and starting from this artificial construction, behind a Gly4Ser linker, a cysteine was introduced again. This should increase the distance of the inserted cysteine from the cysteines in the protein and suppress a shuffling of the cysteines. The resulting artificial construct was finally sequenced and in some titration experiments with the Ellmann reagent it was shown to be suitable for coupling experiments.

For the replacement of cysteine at position 4 with a serine in the AffilinTM SPC1-A7 variant, the QuickChange® PCR method (from Stratagene, La Jolla, USA) was used with primers A7Cys4Ser_for and A7Cys4Ser_rev. For the PCR reaction, 5 μl of a 10 x reaction buffer (100 mM KCl, 100 mM (NH4) 2SO4, 200 mM Tris-HCl, pH 8.8, 20 mM MgSO4, Triton® X-100 al 1%, 1 mg / ml BSA), in each case 125 ng of the two primers, 1 µl of the Pfu-Turbo DNA polymerase, 1 µl of a mixture of dNTP's and H2O until reaching a total volume of 50 µl . The template gene for the Affilin ™ variant SPC1-A7 was served as template DNA in vector pET20b. A first stage of denaturation was started for 3 min at 95 ° C. After this, 18 repeated cycles of denaturation, linker attachment and synthesis followed. Denaturation at 95 ° C lasted 30 s, addition of primers was carried out for 1 min at 60 ° C. The duration of the synthesis step was 5 min at 68 C. At the end of the PCR, a final synthesis was carried out for 5 min at 68 ° C. Amplification was checked by electrophoresis through an agarose gel. A successful amplification was followed by restriction digestion of the template DNA used with the aid of the restriction enzyme DpnI. 1 µl of the enzyme (10 U / µl) was pipetted into the PCR batch, mixed and incubated for 1 hr at 37 ° C. The vector was then introduced by electroporation into a competent strain (X1-1blue, from Stratagene). For this, 1 µl of the DpnI-treated restriction batch was pipetted onto ice to 50 µl of electrocompetent XL-1 blue cells, mixed and pulsed into a chilled (0.1 mm) electroporation cuvette with Ice at 2.5 kV, 25 μF and 200 O. The cells were collected in 1 ml of SOC medium and incubated for 60 min at 37 ° C mediating overcooling at 500 rpm. Cells were then plated onto selection medium (2 x YT, 100 µg / ml ampicillin) and incubated at 37 ° C for 16 hr. From the obtained clones, 12 of them were sequenced separately to control the correct incorporation with the pETTerm primer. The correct clone vector served as template DNA for the following QuickChange® PCR to effect the incorporation at the C-terminus of two glycins, one cysteine and four additional histidines. As described above, QuickChange® PCR, subsequent DpnI digestion and transformation of strain XL1-Blue were carried out with the two primers A7Gly2Cys_for and A7Gly2Cys_rev by electroporation. For the control of correct incorporation, the plasmids of 12 clones were sequenced. The plasmid from a correct clone (SPC-1-A7JJ) was introduced into the expression strain BL21 with the plasmid pUBS520, in order to then express the AffilinTM variant SPC1-A7BB, and as described above ( in Example 1), purify it in two chromatographic steps (affinity chromatography in the presence of Ni-NTA and filtration through a gel in the presence of Sephadex 75).

To check the accessibility (Haber, 1972) of the introduced cysteine residue, all free SH groups should be titrated using Ellmann's reagent (a DTNB solution). For this, to 1 ml of a solution of a protein of the AffilinTM variant SPC1-A7JJ (50 - 350 µg of a protein in 100 mM Tris / HCl; pH 8.0), 30 µl of a solution of DTNB (4mg / ml DTNB in 100mM Tris / HCl, pH 8). 1 ml of the protein solution with 30 μl of a buffer (100 mM Tris / HCl, pH 8) represented the value of vacuum 1. 1 ml of a buffer (100 mM Tris / HCl, of pH 8) with 30 μl of a DTNB solution. The batches were incubated

for 15 min at room temperature and the absorption at 410 nm was measured. The absorptions of vacuum values 1 and 2 were subtracted from the absorption of the test run. The molar concentration of the free SH groups was calculated using the DTNB-SH extinction coefficient using the extinction coefficient (£ 410 [DTNB-SH] =

13,600 M-1cm-1) and was divided by the concentration of the protein used. As a result, the number of free thiol groups was obtained for each protein molecule. In such context, in the case of the constructed variant of Affilin ™ SPC1-A7JJ, 3-4 free cysteine residues could be titrated. A free cysteine residue was expected, since the cysteines present in the protein, as verified when performing the titration control with the Affilin ™ variant SPC-1-A7Cys4Ser, are not accessible. This points to the possibility of a cysteine shuffle of the introduced cysteine at the C-terminus with the cysteines hidden within the protein, and therefore too small a distance from the introduced cysteine to the protein. Because of this, the C-terminus localized cysteine of the AffilinTM variant SPC1-A7JJ was replaced by a serine using a QuickChange® PCR as described above with primers A7Gly2Ser_for and A7Gly2Ser rev. After verifying correct incorporation, this artificial construct served as a template for a PCR to effect incorporation of a Gly4Ser linker followed by a cysteine. Using the primers Gly4SerCys_HindIII and A7Cys4Ser_Nde, a PCR was carried out. For the PCR reaction, 5 µl of the reaction buffer 10 x (100 mM KCl, (100 mM (NH4) 2SO4, 200 mM Tris-HCl, pH 8.8, 20 mM MgSO4, Triton® X-100 at 1 %, 1 mg / ml BSA), in each case 125 ng of both primers, 1 µl of the Pfu-Turbo DNA polymerase, 1 µl of a mixture of dNTP's and H2O until reaching a total volume of 50 µl. Furthermore, for the dissolution of secondary primer structures, 2 µl of DMSO was added to the reaction mixture. Contrary to the procedure described above, 25 repeated steps of denaturation, primer attachment and synthesis were carried out, and at a modified primer attachment temperature of 58 ° C. Amplification of the PCR product of this Affilin ™ variant (SPC1-A7_Cys) was verified by electrophoresis through an agarose gel. Table 2 reproduces a compilation about the artificial constructions described here (see Table 2). A restriction cleavage site for the NdeI enzyme has been integrated into the A7Cys4Ser_Nde primer and a restriction cleavage site for the HindIII enzyme has been integrated into the Gly4SerCys_HindIII primer, making it possible to bind the PCR product after its purification and restriction by means of the two enzymes in the vector pET20b also treated with NdeI and HindIII. The restriction by the two enzymes was carried out at the same time in a double digestion. For this, approximately 1 µg of the PCR product of the AffilinTM variant SPC1-A7_Cys or respectively 1 µg of the vector pET20b were incubated with 1 µl of the restriction enzyme NdeI (from New England Biolabs, Frankfurt am Main, Germany, 20 U / µl) and 1 µl of the restriction enzyme HindIII (from New England Biolabs, Frankfurt am Main, Germany, 20 U / µl) as well as 10 µl of the reaction buffer 10 x NEB 2 (50 mM NaCl, Tris-HCl 10 mM, pH 7.9, 10 mM MgCl2, 1 mM DTT) in a total volume of 100 µl for 4 h at 37 ° C. The resulting fragments were separately purified by preparative electrophoresis through an agarose gel. For the ligation, 20 ng of the fragment of the vector purified and treated with NdeI / HindIII and 120 ng of the fragment treated in the same way of the variant of AffilinTM SPC1-A7Cys were used, as well as 2 µl of the reaction buffer 10 x (Tris-HCl 300mM, pH 7.8, 100mM MgCl2, 100mM DTT, 10mM ATP) and 0.5 µl of T4 DNA ligase (from Promega, Mannheim, Germany 1-3 U / µl) in total volume 20 μl. The reaction batch was incubated for 16 hr at 16 ° C and the resulting vector was introduced as described above into XL-1 blue cells by electroporation. The correct introduction of the gene was controlled by 12 clones obtained after transformation by sequencing with the pETTerm primer. E. coli cells (BL21 (DE3), + pUBS520) were then transformed with the Affilin ™ variant SPC1-A7_Cys vector with a correct sequence. After expression and purification as described for the AffilinTM SPC1-A7 variant (Example 1), for the AffilinTM SPC1-A7_Cys variant, meanwhile, as described above, the residues were titled of cysteine free with the help of Ellmann's reagent. In this case, as expected, only one cysteine residue could be detected, thus confirming the successful incorporation of a cysteine at the C-terminus, accessible to the solvent, at a sufficient distance from the protein for selective coupling to corresponding participants (see Table 1). This detection of an individual accessible cysteine forms the basis for the selective coupling of AffilinTM to suitable coupling partners and matrices.

In order to provide AffilinTM SPU11-3-A1 with a C-terminus cysteine, the gene for SPU11-3-A1 was cloned into the modified pET20b for AffilinTM SPC1-A7 (see above) through the restriction cut sites with NcoI and XhoI. The expression and purification of the Affilin ™ SPU11-3-A1_Cys were carried out identically to the procedure as in the case of SPC1-A7_Cys.

Example 4

Analysis of the binding properties of the AffilinTM variant SPC1-A7_Cys modified with a peptide linker (with a cysteine at the C-terminus)

The binding properties of the Affilin ™ SPC1-A7_Cys purified as in Example 3 were tested in a concentration dependent ELISA. For this, the wells of a NUNC plate were coated with 100 µl of a solution of an antigen (10 µg / ml of the Fc part of a human monoclonal IgG, from Roche) overnight at 4 ° C. The ELISA plate was blocked the following day with PBS (3% BSA, 0.5% Tween 20) for 2 h at room temperature. After washing the wells with PBS (0.1% Tween 20), the modified AffilinTM was added in a concentration dependent manner to the wells (concentration range 10 µM - 0 µM) and incubated for 1 h can. After the wells had been washed again with PBS (0.1% Tween 20), the anti-hGC monoclonal antibody (POD-conjugated; Biogenes, Berlin) was applied in a dilution of

1: 1,000 (50 µl / well) and incubated again for 1 hr at RT. After this, the wells were washed with 3 x PBS (Tween 20 0.1) and 3 x PBS and the color reaction was started with TMB Plus (from Kementec, Denmark) (50 µl / well). After incubating for 20 minutes at room temperature, the color reaction was stopped by adding 0.2M H2SO4 (50 µl / well). The yellow staining obtained was recorded by reading at 450 nm (reference wavelength: 620 nm) and documented (see Fig. 4). Evaluation of the measurement points provided an apparent KD value of 233 mM, roughly corresponding to that of the unmodified Affilin ™ SPC1-A7 and SPC1-A7BB (280 nM). Therefore, modification at the C-terminus of Affilin ™ SPC1-A7 with a peptide linker, including a cysteine, has no impact on the binding capacity of the variant.

Example 5

Selective coupling of AffilinTM SPC1-A7_Cys, which binds to an IgG, with phycoerythrin (PE)

The coupling of the AffilinTM SPC1-A7_Cys, which binds IgG, to an activated PE was carried out as follows:

1 mg / ml SPC1-A7_Cys (in a PBS) was reduced with 10mM DTT for 30 min at room temperature. During the reduction phase, a PD-10 column (from Amersham Biosciences) was rinsed with 5 column volumes of a PBS. After reduction was performed, the reaction mixture was added to the column with equilibrated PD-10, in order to remove excess DTT. The SPC1-A7_Cys reduced in this way was mixed in the molar ratio of 5: 1 with a maleinimide activated phycoerythrin (from Prozyme) and incubated for 1 hr at room temperature with slight shaking. Following this, the unreacted Affilin ™ free sulfhydryl groups were blocked by addition of NEM (N-ethyl-maleinimide) for 20 min at RT. The reaction mixture was then purified by filtration through a gel (Sephadex S-200 HR) and the corresponding fractions were pooled and stored at 4 ° C. The conjugate analysis was carried out by spectroscopy. For this, the absorption spectra in the 250-750 nm range were recorded and the concentrations of PE and Affilin ™ were determined through the extinction coefficients determined or respectively supplied concomitantly. The conjugate obtained in this way based on Affilin ™ SPC1-A7_Cys and PE (SPC1-A7_Cys_PE) was investigated at BIACORE for its binding properties against an IgG-Fc. For this, in the case of a continuous flow rate of 30 µl / min and with HBS-EP as elution buffer, a CM5 chip coupled with IgG-Fc was used. Consecutively, different concentrations of SPC1-A7_Cys-PE were conducted through the chip and the sensorgrams obtained were analyzed with the BIACORE-Evaluation software. In this case, it was shown that an avidity effect was obtained by coupling, which was manifested in a decrease in the macroscopic dissociation constant from KD = 107 M to KD = 10-8 M (see Fig. 5).

Example 6

Nonspecific coupling of Oyster® 556 fluorescent dye to AffilinTM SPC1-A7BB, which binds to an IgG

The Oyster® 556 fluorescent dye (from Molecular Probes) was coupled to the Affilin ™ SPC1-A7BB that binds to an IgG, (without any free cysteine!) And some research was done on the binding.

The coupling process was performed as follows: 1 mg / ml SPC1-A7BB was mixed in a 10 mM phosphate buffer (pH 8.5) in the 1: 2 molar ratio with the fluorescent dye Oyster® 556 ( dissolved in 20 µl dry DMF) and incubated for 30 min at RT. The coupling reaction was stopped by adding 1 volume of a 10% solution of glycine and the batch was purified through a column with PD-10. The degree of coupling was then quantified by spectrometry. For this, the concentration of the conjugate was determined through absorption at 280 nm and corrected with a concomitantly supplied correction factor (from Molecular Probes). The degree of coupling was then obtained from the ratio of the concentrations of Oyster® 556 and the conjugate and could be determined with 1 molecule of AffilinTM / 0.8 molecules of Oyster® 556. The analysis of the binding capacity of the conjugate obtained from this was carried out by means of a concentration-dependent ELISA (for the embodiment see Example 1), as well as by measurements with the BIACORE (see Fig. 6). It could be shown that the binding ability of Affilin ™ SPC1-A7BB is not impaired after coupling with Oyster® fluorescent dye.

Example 7

Nonspecific Coupling of Horseradish Peroxidase (POD) Enzyme to AffilinTM SPCI-A7BB, which Binds to an IgG

The Affilin ™ SPC1-A7BB variant could be unspecifically coupled with the POD enzyme, and binding studies showed preservation of the binding activity of Affilin ™ as well as the enzymatic activity of POD. The conjugate was prepared according to the following protocol:

5 mg of freeze-dried horseradish peroxidase (POD, from Sigma) were dissolved in 250 µl of very pure water, mixed with 37.5 µl of 0.1 M sodium periodate solution, and incubated for 10 min at 20 ° C. After this, 25 µl of ethylene glycol was added and incubated for another 5 min at 20 ° C. The peroxidase was dialyzed by filtration through a gel (G25, column with NAP-5) against pure water.

250 µl of purified AffilinTM SPC1-A7BB (IMAC, gel filtration, 4 mg / ml PBS) was mixed with 100 µl of 0.1M carbonate buffer (pH 9.6) and added 1 mg of activated peroxidase (from Sigma) (approximately 100 μl). The coupling mixture was incubated with shaking for 2 hr at 20 ° C. Then, for each ml of the coupling mixture, 10 µl of 0.5M sodium borohydride was added, mixed briefly and incubated for another 2 h at 4 ° C without shaking. The reaction batch was subjected to a buffer change by PBS with a column with G25. For preservation 0.1% Thiomersal (from Roth) was added. The investigation of the binding activity of the conjugate to human IgG was carried out as follows:

The labeled AffilinTM was diluted in PBS (BSA 0.5%, Tween 20 0.05%, Thiomersal 0.01%) and the solutions were applied to a microtiter plate coated with human IgG (10 mg / ml, 100 ml / ml). The incubation time period was 1 hr at room temperature. The wells were then washed 3 times with in each case 250 ml of a PBS (Tween 20 0.1%, Thiomersal 0.01%) and incubated again with 100 µl TMB for 10-20 min at room temperature. The reaction was terminated by adding 100 µl of 0.5M sulfuric acid. Absorption (at 450nm vs. 620nm reference) was measured on a photometer for microtiter plates (Fig. 7). POD activity could be detected up to a 1: 10,000 dilution of the reaction batch, demonstrating successful coupling of a POD to SPC1-A7BB. By means of control measurements, it was possible to exclude that an uncoupled POD falsifies the signals.

Example 8

Specific and nonspecific coupling of an AffilinTM based on crystalline gamma II and ubiquitin to matrices

AffilinTM coupling to matrices could be done with the following methods:

1.) Coupling of AffilinTM SPU3-A1_Cys through a cysteine located at the C-terminus to a dextran matrix 2.) Coupling of AffilinTM SPC7-E9 through primary amino groups to the dextran matrix of the BIACORE system and 3.) Nonspecific coupling of the Affilin ™ SPC7-E9 with the help of EDC / NHS to a matrix of a polymethacrylate.

1.) The coupling of SPU3-A1_Cys to the dextran matrix of the BIACORE system was carried out selectively through the cysteine introduced at the C-terminus. For this, the carboxyl groups of the dextran matrix were activated with NHS / EDC with a contact time period of 2 min and then mixed with the thiol coupling reagent PDEA (2- (2-pyridinyl-dithio) ethanamine, in a 0.1 M borate buffer, pH 8.5) . After a reaction time period of 4 min, a purified SPU3-A1_Cys (in a 20 mM phosphate buffer, pH 6.0) was added to the dextran chip thus modified and the reaction continued for 7 min. Deactivation of the chemically unconverted PDEA groups was carried out with 50mM L-cysteine (1M NaCl) for 4 min. With this method, 350 units (RU) of SPU3-A1_Cys could be immobilized on the chip and used for other kinetic analyzes. After kinetic measurements, the chip was regenerated with 0.1 glycine (pH 2.2), 6M Gua / HCl, 6M urea and 20% ethanol. Affilin ™ chip binding affinity remained conserved after 20-30 regeneration cycles as well (see Fig. 8).

2.) Furthermore, SPC7-E9 could be unspecifically coupled through amino groups (lysines) exposed on the surface with NHS / EDC to the carboxyl groups of the dextran matrix of BIACORE in the following way: The CM5 chip it was activated for 7 min with NHS / EDC and then a purified SPC7-E9 (in a 20 mM Na phosphate buffer, pH 6.0) was driven through the chip for another 7 min. After coupling was performed, the remaining reactive groups were quenched with 1M ethanolamine. For analysis of dissociation constants, the proNGF target was driven through the chip at different concentrations, the binding monitored online (see Fig. 9) and the curves were then evaluated with the BiaEvaluation software. The KD value could thus be determined as 1.4 nM. After kinetic measurements, the chip was regenerated with 0.1 glycine (pH 2.2), 10mM HCl, 10mM NaOH, 6M Gua / HCl, 6M urea and 20% ethanol. The binding activity of the chip with Affilin ™ remained also after several regeneration cycles.

3.) The purified SPC7-E9 protein (4 mg) was subjected to a buffer change through a column with PD-10 (from Amersham) in a 0.1 M borate buffer (0.5 M Na2SO4, pH 9) and coupled to Fractogel® EMD Epoxy (M). For this, the gel (0.5 g) was incubated in a 0.1 M borate buffer (0.5 M Na2SO4, pH 9) for 2 h at RT and after this was washed several times with this buffer. The coupling reaction was started by adding SPC7-E9 to the epoxy matrix and continued at RT for 24 h with continuous stirring. A reference column

without SPC7-E9 it was also provided as a control and was treated identically. Using 1 M ethanolamine (pH 9.5), the reaction was stopped for 48 h at RT and the gel matrix was washed with a buffer of sodium acetate (0.1 M, pH 4.0), NaCl 1 M and a PBS (in each case 50 column volumes). The Affilin ™ SPC7-E9 affinity matrix produced in this way was column filled with C (from Amersham Biosciences, 1 x 10 cm) and connected to a chromatography facility (Äkta Explorer, from Amersham Biosciences). As the elution buffer, a PBS (0.5 mM EDTA) was used in all cases at a circulation rate of 1 ml / min. For the assay of column binding ability with Affilin ™ produced in this way, a purified proNGF was applied. After rinsing the column with 10-20 column volumes of the elution buffer, the fixed proNGF was eluted with 0.1M glycine (pH 2.2) (see Fig. 10). The separation of proNGF was further achieved from mixtures of substances with BSA and from a crude extract of E.coli. For this, 1 ml of the BSA solution (5 mg / ml, from Sigma) was mixed with 0.5 ml of proNGF (1.3 mg / ml) and applied to the column with Affilin ™. After rinsing the column with 20 column volumes of the elution buffer, the unbound proNGF was eluted with glycine (0.1M, pH 2.2). After a column regeneration with 10 column volumes of 6M Gua / HCl, a mixture of 1 ml of a crude extract of E.coli (a resulting soluble supernatant material after the Disintegration (with lysozyme / benzonase / ultrasound) of the bacteria pellet from 50 ml of an overnight culture of B121) and 0.5 ml of proNGF (1.3 mg / ml) on the column with Affilin ™. After rinsing the column with 20 column volumes of the elution buffer, the unbound proNGF was also eluted with glycine (0.1M, pH 2.2). The column was then regenerated with 0.1M glycine (pH 2.2), 10mM HCl, 10mM NaOH, 6M Gua / HCl, 6M urea and 20% ethanol. Fractions eluted from separation of proNGF from BSA and crude E. coli extract were analyzed by electrophoresis through an agarose gel (see Fig. 11). After 10 test courses an unmodified binding of proNGF to the column could be observed with SPC7-E9.

Annexed

Table 1:

Variant A410 Vacuum value Vacuum value A410 corr. Concentration Number of Cysteines Number1 2 used cysteines in the accessible of [µg / ml] protein theoretically cysteines labeled

SPC-1-A7BB 0.2142 0.08970.1232 0.0013 507 0 0 0.2254 0.1003 0.1232 0.0019 100

SCP-1-A7JJ 0.3154 0.08320.1232 0.109508 1 4 0.4412 0.0738 0.1232 0.2442 100

SPC-1-A7-Cys 0.235 0.08340.1232 0.0286 508 1 0.272 0.0966 0.1232 0.0522 10

Table 2:

DNA sequence of the human crystalline gamma II library

AffilinTM DNA Sequences Based on Crystalline Gamma II

SPC1-A1 (SEQ ID NO: 2)

SPC1-A7 (SEQ ID NO: 3)

SPC1-G3 (SEQ ID NO: 4)

SPC1-A7JJ (including His10) (SEQ ID NO: 6)

SPC1-A7_Cys (including His10) (SEQ ID NO: 7)

Modified Ubiquitin (MUBI) (SEQ ID NO: 10)

UB10 (library) (SEQ ID NO: 11)

AffilinTM DNA Sequences Based on Ubiquitin

SPU11-3-A1_Cys (including His10) (SEQ ID NO: 13)

Primer:

SEQ ID NO: 25

Bibliography cited:

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10 structure and stability of the eye lens: x-ray analysis of gamma-crystallin II. Nature 289, 771-777. Buchberger A, Howard MJ, Proctor M, Bycroft M, National Library of Medicine, J Mol Bil. 2001 Mr 16; 307 (1); 17-24. Butt, T. R., Jonnalagadda, S., Monia, B. P., Sternberg, E. J., Marsh, J. A., Stadel, J. M., Ecker, D. J. and Crooke,

S. T. (1989) Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli. PNAS 86, 2540-2544. Finucane, M. D., Tuna, M., Lees, J. H., and Woolfson, D. N. (1999). Core-directed protein design. I. An experimental

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Hanes, J. et al. (1997): In vitro selection and evolution of functional proteins by using ribosome display. Proc. Natl. Acad. Sci. USA. 94, 4937-42. Hazes, B. and Hol, W. G. J. (1992): Comparison of the hemocyanin �-barrel with other greek key �-barrels: possible importance of the "�-zipper" in protein structure and folding. Proteins: Struct., Funct. Gen. 12, 278-298. Hemmingsen, J. M., Gernert, K. M., Richardson, J. S. and Richardson, D. C. (1994): The tyrosine corner: a feature of most greek key �-barrel proteins. Prot. Science 3, 1927-1937. Herrmann, J. E., and Morse, S. A. (1973) Coupling of peroxidase to poliovirus antibody: Characteristics of the conjugates and their use in virus detection. Infection and Immunity, 645-649. Jaenicke, R. (1994). Eye-lens proteins: structure, superstructure, stability, genetics. Naturwissenschaften 81, 423

429. Jaenicke, R. (1996). Stability and folding of ultrastable proteins: eye lens crystallins and enzymes from thermophiles. Phase J 10, 84-92. Jaenicke, R., and Slingsby, C. (2001). Lens crystallins and their microbial homologs: structure, stability, and function. Crit Rev Biochem Mol Biol 36, 435-499. Kumaraswamy, V. S., Lindley, P. F., Slingsby, C., and Glover, I. D. (1996). An eye lens protein-water structure: 1.2 angstrom resolution structure of gamma B-crystallin at 150K. Acta Crystallogr D Biol Crystallogr 52, 611. Larsen CN, Wang H., National Library of Medicine; J Proteome Res. 2002 Sep-Oct; 1 (5): 411-9. Lazar, G. A., Desjarlais, J. R., and Handel, T. M. (1997). De novo design of the hydrophobic core of ubiquitin. Protein Sci 6, 1167-1178. Ling, M. M. (2003). Large antibody display libraries for isolation of high-affinity antibodies. Comb Chem High Throughput Screen 6, 421-432. Lottspeich, F., and Zorbas, H. (1998). Bioanalytik (Heidelberg, Spektrum Akademischer Verlag). Mandal, K., Chakrabart, B., Thomson, J. and Siezen, R. J. (1987): Structure and stability of �-crystallins. Denaturation and proteolysis behavior. J. Biol. Chem. 262, 8096-8102. Mayr, E. M., Jaenicke, R., and Glockshuber, R. (1994). Domain interactions and connecting peptides in lens crystallins. J Mol Biol 235, 84-88. Murzin A. G., Brenner S. E., Hubbard T., and Chothia C. (1995). SCOP: a structural classification of proteins database for the investigation of sequences and structures. J. Mol. Biol. 247, 536-540. Najmudin, S., Nalini, V., Driessen, H. P., Slingsby, C., Blundell, T., Moss, D., and Lindley, P. (1993). Structure of bovine gB (g II) -crystalline at 1.47 Ä. Acta Crystallogr D Biol Crystallogr 49, 223-233. Norledge, B. V., Mayr, E. M., Glockshuber, R., Bateman, O. A., Slingsby, C., Jaenicke, R., and Driessen, H. P. (1996). The X-ray structures of two mutant crystallin domains shed light on the evolution of multi-domain proteins. Nat Struct Biol 3, 267-274. Reichlin, M. (1980) Use of glutaraldehyde as a coupling agent for proteins and peptides. Methods Enzymol 70, 159

165. Richardson, J. S., Richardson, D. C., Tweedy, N. B., Gernert, K. M., Quinn, T. P., Hecht, M. H., Erickson, B. W., Yan, Y., McClain, R. D., Donlan, M. E. and Surles, M. C. (1992): Looking at proteins: representations, folding, packing and design. Biophys. J. 63, 1186-1209. Riddle, D. S., Santiago, J. V., Bray-Hall, S. T., Doshi, N., Grantcharova, Q. Y and Baker, D. (1997): Functional rapidly folding proteins from simplified amino acid sequences. Nature structural biology 4, 805-809. Rudolph, R., Siebendritt, R., Nesslauer, G., Sharma, A. K., and Jaenicke, R. (1990). Folding of an all-beta protein: independent domain folding in gamma II-crystallin from calf eye lens. Proc Natl Acad Sci U S A 87, 4625-4629. Sambrook, J., Maniatis, T. and Fritsch, E.F. (1989): Molecular Cloning: A laboratory manual. Cold spring harbor. Cold Spring Harbor Laboratory Press, New York. Sharma, A. K., Minke-Gogl, V., Gohl, P., Siebendritt, R., Jaenicke, R., and Rudolph, R. (1990). Limited proteolysis of gamma II-crystallin from calf eye lens. Physicochemical studies on the N-terminal domain and the intact twodomain protein. Eur J Biochem 194, 603-609. Slingsby, C. (1985): Structural variation in lens crystallins. TIBS 10, 281-284. Smith, G. P (1985): Filamentous Fusion Phage: Novel expression vectors that display cloned antigens on the virion surface. Science 228, 1315-1317. Stahl, S. and Uhlen, M. (1997): Bacterial surface display: trends and progress. TIBTECH 15, 185-192. Takamiya, H., Shimizu, F., and Vogt, A. (1975) A two-stage method for cross-linking antibody globulin to ferritin by glutaraldehyde.III. Size and antibody activity of the conjugates. J Immunol Methods 8 (4), 301-306. Vijay-Kumar, S., Bugg, C. E., and Cook, W. J. (1987). Structure of ubiquitin refined at 1.8 A resolution. J Mol Biol 194, 531-544. Voet, D., and Voet, J. G. (1995). Biochemistry, Second Edition edn (New York, John Wiley & Sons, Inc.). Wistow, G., Turnell, B., Summers, L., Slingsby, C., Moss, D., Miller, L., Lindley, P., and Blundell, T. (1983). X ray analysis of the eye lens protein gamma-II crystallin at 1.9 A resolution. J Mol Biol 170, 175-202. Wistow, G. J., and Piatigorsky, J. (1988). Lens crystallins: the evolution and expression of proteins for a highly specialized tissue. Annu Rev Biochem 57, 479-504.

LIST OF SEQUENCES

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<213> Artificial

<220>

<223> Mold

<400> 28 gtcaaagcta aaatccaaga caaagaagga attccacctg accagcaacg cct

<210> 29

<211> 50

<212> DNA

<213> Artificial

<220>

<223> Mold

<400> 29 gggtgagccc gtcctctagt tgtcgtcctg cgaaagctag gcgttgctgg 50

<210> 30

<211> 50

<212> DNA

<213> Artificial

<220>

<223> Mold

<400> 30 gacgggctca ccctgtctga ctacaacatc caaaaagaat ccaccctcca 50

<210> 31

<211> 39

<212> DNA

<213> Artificial

<220>

<223> Mold

<400> 31 gagtgctcgc agcaggagtg ccaggtggag ggtggattc 39

<210> 32

<211> 22

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 32 gatatacata tgcaaatctt cg 22

<210> 33

<211> 18

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 33 gtggtgctcg agtgctcg 18

<210> 34

<211> 55

<212> DNA

<213> Artificial

<220>

<223> primer

<220>

<221> Varied feature

<222> (20) .. (21)

<223> n is a, c, g, or t

<220>

<221> Varied feature

<222> (26) .. (27)

<223> n is a, c, g, or t

<220>

<221> Varied feature

<222> (32) .. (33)

<223> n is a, c, g, or t

<400> 34 ccagccggcc atggccatgn nkatcnnkgt tnnkaccctg acgggaaaga ctatc

<210> 35

<211> 55

<212> DNA

<213> Artificial

<220>

<223> primer

<220>

<221> Varied feature

<222> (23) .. (24)

<223> n is a, c, g, or t

<220>

<221> Varied feature

<222> (26) .. (27)

<223> n is a, c, g, or t

<220>

<221> Varied feature

<222> (29) .. (30)

<223> n is a, c, g, or t

<220>

<221> Varied feature

<222> (32) .. (33)

<223> n is a, c, g, or t

<220>

<221> Varied feature

<222> (35) .. (36)

<223> n is a, c, g, or t

<400> 35 caggaggagt gccaggtgga gmnnmnnmnn mnnmnngatg ttgtagtcag acagg 55

<210> 36

<211> 34

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 36 gttattactc gcggcccagc cggccatggc catg 34

<210> 37

<211> 48

<212> DNA

<213> Artificial

<220>

<223> primer

<400> 37 gagtttttgt tcggcctcga gggcccgcag gaggagtgcc aggtggag 48

Claims (18)

1. Procedure for the preparation of a conjugate, which includes the following components: one or more polypeptide molecules (I) based on human ubiquitin, and, covalently bound with it, one or various functional components (II) that are chosen from the set consisting of polypeptides and proteins, organic polymers, sugars, low molecular weight substances, peptides, as well as derivatives of these substances, realizing that, after the coupling of (I) to (II), the functionality of all components remains preserved, and realizing that the procedure comprises the following steps: a) Provision of human ubiquitin; b) Provision of a ligand, which is chosen from the set consisting of some (poly) peptides / proteins; c) Selection of amino acids 2, 4, 6, 62, 63, 64, 65 and 66 of human ubiquitin; d) Modification of the selected amino acids by substitution mediating conservation of the motif of ubiquitin-type folding; e) Contacting the modified protein with the ligand made available in step b); f) Determination of those proteins, which are suitable for a specific binding to ligands with a dissociation constant of KD = 10-5 M or less, detection of binding activity being performed at using the ELISA technique or a superficial plasmon resonance; g) Coupling of components (I) and (I) in a region outside the exposed region superficially of the folded sheet � of the polypeptide molecule (I), which is intended for fixation specific for the ligand, through a terminal fusion of peptides with (I), or through side chains cysteine or lysine in (I); h) Isolation of the conjugate; and i) Detection of the functionality of both components of the conjugate. 2. Procedure for the preparation of a conjugate as defined in claim 1, starting from a component (I) with a known sequence, which includes the following steps:

-

Identification of suitable amino acid residues for coupling by analysis of the spatial structure of the protein, preferably of residues located outside the interaction surface of (I) with the ligand;

-

Activation of a partner in the coupling by a suitable coupling reagent;

-

Carrying out the coupling reaction:

-

Isolation of the conjugate; and

-

Detection of the functionality of both components of the conjugate.

3. Procedure for the preparation of a conjugate as defined in claim 1, starting from a component (I) with a known sequence, in which no suitable amino acid residue for coupling had been identified, covering the next stages:

-

Introduction of amino acid residues suitable for coupling by substitution, insertion or fusion, preferably of residues exposed on the surface outside the interaction surface of (I) with the ligands;

-

Detection of the accessibility of the introduced amino acid residues;

-

Detection of the functionality of component (I) modified in this way;

-

Activation of a partner in the coupling by means of a suitable coupling reagent;

-

Carrying out the coupling reaction;

-

Isolation of the conjugate; and

-

Detection of the functionality of the components of the conjugate.

Four.

 Method according to claim 1, wherein the coupling is carried out through the lysine residues 11, 29, 33 and 48 of the ubiquitin molecule.

5.

 Method according to claim 2, it being realized that the additional terminal fusion of peptides with (I) contains one or more cysteine residues or one or more lysine residues, being realized that these amino acid residues do not preferably participate in the interaction of (I ) with the ligand.

6.

 Method according to one or more of claims 1-5, wherein the functional component is realized

(II) is a peptide, a polypeptide or a protein, preferably a chromophore protein, an enzyme, an immunoglobulin, an immunoglobulin derivative, a toxin or a polypeptide according to I. 7. Method according to one or more of claims 1-5, wherein the functional component is realized (II) is a polymer, preferably a dextran, a polymethacrylate, a Sepharose, an agarose, a vinyl polymer, a polystyrene, a silica gel, a cellulose, a poly (ethylene glycol) or a derivative of a polymer. 8. Method according to one or more of claims 1-5, wherein the functional component is realized (II) is a low molecular weight substance, preferably a dye, biotin, digoxigenin, a heavy metal, a chelating agent, a radioisotope, an antibiotic or a cytotoxic substance. 9. Method according to one or more of the preceding claims, wherein the component

(I)

 has a new binding property generated for specific binding to a ligand, which is a polypeptide

or a protein, preferably immunoglobulins and immunoglobulin derivatives, proteins obtained from blood plasma, factors and inhibitors of blood coagulation, interleukins, cytokines, receptor proteins, glycoproteins, viral proteins and cell surface markers, preferably CD 14, CD 25, CD 34.

10.

Method according to one or more of the preceding claims, it being realized that component (I) has a newly generated binding property for specific binding to a ligand, which is a peptide, preferably an affinity appendage, so preferred an S-brand, His-brand, Strep-brand, Myc-brand or FLAG-brand, or viral peptides.

eleven.

Method according to one or more of the preceding claims, it being realized that component (I) has a new binding property for specific binding to a ligand, which is a low molecular weight substance, preferably steroids, cholesterol and polluting substances, such as, for example, halogenated hydrocarbons.

12.

Method according to one or more of the preceding claims, wherein component (II) is one or more ubiquitin-based polypeptides, which are identical to (I), and are linked by covalent bonds with it, whereby an increase in affinity for the ligands of (I) is achieved through avidity effects.

13.

Method according to one or more of the preceding claims, wherein component (II) is performed as a polypeptide, a protein or a polymer, with which component (I) is linked multiple times by covalent bonds, whereby an increase in affinity for ligands of (I) is achieved through avidity effects.

14.

Method according to one or more of the preceding claims, whereby component (II) is a polypeptide or a polymer, which, after being covalently linked with component (I), begins to take part of covalent bonds or not covalent with other conjugates of this type, with which an increase in the affinity for the ligands of (I) is achieved through avidity effects.

fifteen.

Method according to one or more of the preceding claims, with component (I) being carried out as SPU11-3-A1 (SEQ ID NO: 12 and 13).

16.

 Method according to one or more of the preceding claims, further encompassing the production of a diagnostic kit with a conjugate prepared according to one or more of claims 1-15.

17.

 Process according to one or more of the preceding claims, further encompassing the preparation of a pharmaceutical composition with a conjugate prepared according to one or more of claims 1-15.

18.

 Process according to one or more of the preceding claims, further encompassing the preparation of a composition for affinity enrichment with a conjugate prepared according to one or more of claims 1-15, the functional component being realized to be a membrane , a polymer balls or a chromatographic support material.

Figure 1: Figure 2: Figure 3: Figure 4: Figure 6: Figure 8: Figure 10: Figure 11: